Methods for modulating eicosanoid metabolism

ABSTRACT

The inventive subject matter relates to methods for modulating an eicosanoid metabolic process in cells of an animal in need thereof, which comprises administering to the animal an amount of the inventive compositions effective for regulating the activity of an eicosanoid oxygenase. In particular, the inventive subject matter relates to methods for modulating arachidonic acid metabolism by administering an amount of the inventive compositions effective for regulating the activity of lipoxygenases and cyclooxygenases.

This application is a continuation of U.S. patent application Ser. No.11/452,246, filed Jun. 14, 2006, the contents of which is incorporatedherein by reference. This application claims the benefit of U.S.Provisional Patent Application Nos. 60/690,161, filed Jun. 14, 2005, andNo. 60/792,330, filed Apr. 17, 2006, the contents of which are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTIVE SUBJECT MATTER

1. Field of Inventive Subject Matter

The inventive subject matter relates to methods for modulating aneicosanoid metabolic process in cells of an animal in need thereof,which comprises administering to the animal an amount of the inventivecompositions effective for regulating the activity of an eicosanoidoxygenase. In particular, the inventive subject matter relates tomethods for modulating arachidonic acid metabolism by administering anamount of the inventive compositions effective for regulating theactivity of lipoxygenases and cyclooxygenases.

2. Background

Although progress has been made in the early diagnosis and treatment ofcancers such as prostate cancer, prostate cancer remains the most commonmalignancy and the second leading cause of male cancer related deaths inthe United States. One of the more interesting aspects of this diseaseis the fact that latent prostate cancer occurs at equal rates in bothAsian and American men, while the incidence of clinically significantprostate cancer is much greater in the US than in Asia. There are manyreasons to believe that this discrepancy is related to the dietaryintake of different populations and these observations have stimulatedextensive research into various dietary factors that might influenceprogression of prostate cancer. Some epidemiological studies suggestthis may be in part due to the lower fat intake in Asian diets comparedto the typical Western diet, as high fat diets have been linked toelevated risks of prostate cancer.

Applicants have found that administration of the inventive compositionsinhibits TNF-induced NF-κB activation in myeloid leukemia KBM-5 cellsand cigarette smoke condensate-induced NF-κB activation in lungadenocarcinoma H1299 cells. Administration of the inventive compositionsalso leads to suppression of TNF-induced cell invasion and abrogatedRANKL-induced osteoclastogenesis. Further, administration of theinventive compositions suppresses TNF-induced expression of variousantiapoptotic, proliferative, and metastasis gene products.

SUMMARY OF THE INVENTIVE SUBJECT MATTER

The inventive subject matter relates to a method for modulating aneicosanoid metabolic process in cells of an animal in need thereof,which comprises administering to the animal an amount of a compositioneffective for regulating the activity of an eicosanoid oxygenase,

-   -   wherein the composition comprises therapeutically effective        amounts of supercritical extracts of rosemary, turmeric, oregano        and ginger; and therapeutically effective amounts of        hydroalcoholic extracts of holy basil, ginger, turmeric,        Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese        goldthread, and barberry.

The inventive subject matter further relates to a method of delivering13-S-HODE to an animal in need thereof, comprising administering to theanimal a composition comprising therapeutically effective amounts ofsupercritical extracts of rosemary, turmeric, oregano and ginger; andtherapeutically effective amounts of hydroalcoholic extracts of holybasil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea,huzhang, Chinese goldthread, and barberry.

Additionally, the inventive subject matter relates to a method forinhibiting arachidonic acid-mediated inflammation in an animal in needthereof, comprising administering to the animal a composition comprisingtherapeutically effective amounts of supercritical extracts of rosemary,turmeric, oregano and ginger; and therapeutically effective amounts ofhydroalcoholic extracts of holy basil, ginger, turmeric, Scutellariabaicalensis, rosemary, green tea, huzhang, Chinese goldthread, andbarberry.

Furthermore, the inventive subject matter relates to a method formodulating the level of NF-κB-regulated gene products in cells of ananimal in need thereof, comprising administering to the animal acomposition comprising therapeutically effective amounts ofsupercritical extracts of rosemary, turmeric, oregano and ginger; andtherapeutically effective amounts of hydroalcoholic extracts of holybasil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea,huzhang, Chinese goldthread, and barberry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the inhibitory effect of the compositions ofthe present inventive subject matter on growth of human A549 lung cancerand human PC3 prostate cancer cell lines.

FIG. 2 is a graph depicting the effect of the inventive compositions onformation of PGE2.

FIG. 3 is a graph depicting effect of the inventive compositions onformation of PGE2 and 5-HETE in PC3 cells.

FIG. 4 is a graph depicting the concentration-dependent effect of theinventive compositions on eicosanoid metabolism in human A549 lungcancer cells.

FIG. 5 shows mass spectra data showing presence of 13-S-HODE in theinventive compositions.

FIG. 6 is a graph depicting the anti-inflammatory effect of theinventive compounds on mouse ear edema.

FIG. 7 is a graph depicting the effect of 13-S-HODE on proliferation ofvarious cancer cell lines.

FIG. 8 is a graph depicting the inhibitory effect of the inventivecompounds on growth of human breast cancer MCF7 and MDA231 cell lines.

FIG. 9 is a Western blot depicting the ability of the inventivecompositions to down-regulate in a concentration-dependent manner thepresence of 5-lipoxygenase in human tumor cell lines (e.g. PC3).

FIG. 10 is a graph depicting the effect of the inventive compositions onarachidonic acid (AA) mediated mouse ear edema.

FIG. 11(A) is a series of photographs depicting TRAP-positive RAW 264.7cells following incubation either alone or in the presence of 5 nM RANKLwith 0.8 mg/ml of the inventive composition for 5 days.

FIG. 11(B) is a graph depicting the number of multinucleated (threenuclei) osteoclasts counted in RAW 264.7 cells following incubationeither alone or in the presence of 5 nM RANKL with 0.8 mg/ml of theinventive compositions for 5 days.

FIG. 12 is a graph depicting an invasion assay of a Matrigel invasionchamber after overnight coincubation of 0.3 mg/ml of the inventivecompositions and 1 nM TNF for 24 h in the presence and in the absence of1% serum.

FIG. 13 is a series of photographs depicting cell death of humanmultiple myeloma U266 cells incubated with 1 nM TNF, 1 nM taxol, and 300nM doxorubicin, alone or in combination with 0.5 mg/ml of the inventivecompositions, determined by calcein AM based LIVE/DEAD assay asdescribed herein. Red color highlights dead cells, and green colorhighlights live cells.

FIG. 14(A) is a photograph depicting NF-κB activation of KBM-5 cellspreincubated with indicated concentrations of the inventive compositionsand treated with 0.1 nM TNF for 30 min.

FIG. 14(B) is a photograph depicting NF-κB activation of KBM-5 cellspreincubated with 1 mg/ml of the inventive compositions and treated with0.1 nM TNF for 30 min.

FIG. 14(C) is a photograph depicting NF-κB activation of KBM-5 cellspreincubated with 1 mg/ml of the inventive compositions and treated withcigarette smoke (10 μg/ml) for 1 h.

FIG. 15(A) is a photograph depicting the expression of proliferative andmetastatic proteins in KBM-5 cells incubated with 1 mg/ml of theinventive compositions for 1 h and treated with 1 nM TNF for theindicated times.

FIG. 15(B) is a photograph depicting the expression of anti-apoptoticproteins in KBM-5 cells incubated with 1 mg/ml of the inventivecompositions for 1 h and treated with 1 nM TNF for the indicated times.

FIG. 16(A) is a series of graphs depicting cell cycle distributionplots. The upper left plot depicts a normal cell cycle distribution ofPC3 cells. In the upper right plot, the size of the G2/M peak increases.The plot in the lower left and in the lower right show an increasingcell cycle block due to increased concentration of the inventivecompositions.

FIG. 16(B) is a graph depicting the data from the flow cytometryanalyses in FIG. 16(A) as a histogram.

FIGS. 17(A)-17(D) are a series of graphs depicting PC3 cells treatedwith the inventive compositions at indicated concentrations and thenstained with Annexin V and PI solution.

FIG. 17(E) is a graph depicting the data from the flow cytometryanalyses in FIGS. 17(A)-17(D) as a histogram.

FIG. 18(A) is a graph depicting the formation of PGE₂ in relation to theconcentrations of the inventive compositions shown.

FIG. 18(B) is a graph depicting the formation of 5-HETE in relation tothe concentrations of the inventive compositions shown.

FIG. 18(C) is a graph depicting the formation of 12-HETE in relation tothe concentrations of the inventive compositions shown.

FIG. 18(D) is a photograph of a Western blot depicting treatment of PC3cells with the inventive compositions and the cellular content of 5-LOXprotein.

FIG. 19(A) is a photograph of a Western blot depicting retinoblastomaprotein as both unphosphorylated (Rb) and phosphorylated (pRb) proteins.

FIG. 19(B) is a graph depicting the data in FIG. 19(A) after scanningthe gels in a photometric scanning device to permit quantification ofthe density of the bands.

FIG. 20(A) is a graph depicting PC3 cells treated with the inventivecompositions at the indicated concentrations and the inhibition of cellproliferation.

FIG. 20(B), is a photograph of a Western blot depicting a concentrationdependent decline in expression of pRb produced by the inventivecompositions.

DETAILED DESCRIPTION OF THE INVENTIVE SUBJECT MATTER Definitions

The term “modulating” refers to the process of producing an effect onbiological activity, function, health, or condition of an organism inwhich such biological activity, function, health, or condition ismaintained, enhanced, diminished, or treated in a manner which isconsistent with the general health and well-being of the organism.

The term “enhancing” the biological activity, function, health, orcondition of an organism refers to the process of augmenting,fortifying, strengthening, or improving.

The term “eicosanoid” refers to any of the class of compounds derivedfrom polyunsaturated fatty acids, such as arachidonic acid and linolinicacid, and involved in cellular activity.

The term “oxygenase” refers to any of the class of enzymes that catalyzethe incorporation of molecular oxygen into its substrate.

The term “supercritical gas” or “supercritical fluid” as used hereinrefers to a gas is that heated to a temperature critical point, overwhich the gas will maintain its gaseous state and not turn to a liquidregardless of pressure. A gas heated to a temperature above its criticalpoint will become very dense on compression, so that its characteristicsresemble those of a fluid, but will become liquid. Carbon dioxide iscommonly used in applications requiring a supercritical fluid. Thegeneral properties of supercritical fluids and the general use ofsupercritical fluids in extraction processes are described in, e.g.Taylor, Supercritical Fluid Extraction, Wiley, 1996; McHugh andKrukonis, Supercritical Fluid Extraction: Principles and Practice, 2nded., Butterworth-Heinemann, 1994; and Williams and Clifford,Supercritical Fluid Methods and Protocols, Humana Press, 2000, thecontents of which are incorporated by reference herein.

Applicants have developed a mixture comprised of herbal extracts, andthe mixture has COX-2 inhibitory activity. Applicants' compositions areunique, in that some components of the composition are prepared via asupercritical CO₂ extraction process. Unlike traditional solvent basedextraction methods, supercritical CO₂ extraction allows the naturalproducts in the herbs to be obtained without leaving chemical residuesbehind in the preparation.

The term “supercritical extraction” as used herein refers to thetechnique in which hydrophobic compounds can be extracted from samplesutilizing a supercritical fluid. The solvation power of a supercriticalfluid is increased as the pressure and temperature are increased abovetheir critical points, producing an effective solvent for the isolationof hydrophobic molecules. The term “supercritical extracts” refers toextracts prepared by supercritical extraction.

The term “hydroalcoholic extraction” as used herein refers to thetechnique in which hydrophilic compounds can be extracted from a sampleutilizing a solution of alcohol and water, followed by evaporation ofthe solution to produce a extract consisting of dissolved solids. Theterm “hydroalcoholic extracts” refers to extracts prepared byhydroalcoholic extraction.

The term “13-S-HODE” refers to 13-hydroxyoctadeca-9Z, 11E-dienoic acid.

The term “NF-κB” or “nuclear factor kappa B” refers to a multisubunittranscription factor involved in gene expression. Active NF-κB consistsof a dimer of a REL family/p65 subunit and a p50 or p52 subunit. NF-□Bis maintained in the cytoplasm through interactions with its inhibitorIκB, but upon dissociation translocates into the nucleus.

The Inventive Compositions

The inventive compositions are a genus of polyherbal preparationscomprising constituents which exhibit anti-proliferative,anti-inflammatory, antioxidant, anti-angiogenic, and apoptoticactivities. The inventive compositions are comprised of therapeuticallyeffective amounts of supercritical extracts of rosemary, turmeric,oregano and ginger; and therapeutically effective amounts ofhydroalcoholic extracts of holy basil, ginger, turmeric, Scutellariabaicalensis, rosemary, green tea, huzhang, Chinese goldthread, andbarberry.

In one aspect, the active composition comprises:

(A) from about 4.5% to about 7.5%, and more preferably from about 5.5%to about 6.5%, by weight of the hydroalcoholic extract of ginger;

(B) from about 5.5% to about 8.5%, and more preferably from about 6% toabout 8%, by weight of the supercritical extract of ginger;

(C) from about 1.0% to about 1.5%, and more preferably from about 1.2%to about 1.4%, by weight of the supercritical extract of turmeric;

(D) from about 10.0% to about 16.0%, and more preferably from about11.5% to about 14.5%, by weight of the supercritical extract ofrosemary;

(E) from about 4.0% to about 6.0%, and more preferably from about 4.5%to about 5.5%, by weight of the supercritical extract of oregano;

(F) from about 10.0% to about 16.0%, and more preferably from about11.5% to about 14.5%, by weight of the hydroalcoholic extract ofturmeric;

(G) from about 5.5% to about 8.0%, and more preferably from about 6.0%to about 7.0%, by weight of the hydroalcoholic extract of rosemary;

(H) from about 10.0% to about 16.0%, and more preferably from about11.5% to about 14.5%, by weight of the hydroalcoholic extract of holybasil;

(I) from about 10.0% to about 16.0%, and more preferably from about11.5% to about 14.5%, by weight of the hydroalcoholic extract of greentea;

(J) from about 8.0% to about 12.0%, and more preferably from about 9.0%to about 11.0%, by weight of the hydroalcoholic extract of huzhang;

(K) from about 4.0% to about 6.0%, and more preferably from about 4.5%to about 5.5%, by weight of the hydroalcoholic extract of Chinesegoldthread;

(L) from about 4.0% to about 6.0%, and more preferably from about 4.5%to about 5.5%, by weight of the hydroalcoholic extract of barberry; and

(M) from about 2.0% to about 3.0%, and more preferably from about 2.25%to about 2.75%, by weight of the hydroalcoholic extract of Scutellariabaicalensis.

Optionally, the hydroalcoholic extract of ginger, rosemary, turmeric, ororegano used in the present invention is preferably prepared as follows.The plant or a part thereof, which is preferably cryogenically ground topreserve heat sensitive components, is subjected to supercriticalextraction, preferably with carbon dioxide, to obtain an oil extract,referred to herein as “the supercritical extract”. In an additionaloptional embodiment, an oil-free residue is isolated from the first stepand is then extracted in a water/alcohol, preferably water/ethanol,mixture composed of 60-80 parts alcohol and 40-20 parts water. Thealcohol/water liquid is then evaporated off, leaving a powdered extractresidue, referred to herein as “the hydroalcoholic extract”.

Supercritical extracts of ginger, rosemary, turmeric and oreganooptionally used in the present invention can be prepared according toknown supercritical extraction methods, such as disclosed, e.g., in E.Stahl, K. W. Quirin, D. Gerard, Dense Gases for Extraction and Refining,Springer Verlag 4 1988, which is hereby incorporated by referenceherein.

In a preferred aspect, the weight ratio of the supercritical extract ofginger to the hydroalcoholic extract of ginger is from about 0.8:1 toabout 1.4:1.

The hydroalcoholic extracts of rosemary, turmeric, holy basil, greentea, huzhang, Chinese goldthread, barberry and Scutellaria baicalensisused in the present invention can be prepared according to conventionalhydroalcoholic extraction techniques. For example, the hydroalcoholicextracts can be prepared by extracting the plant portion in awater/alcohol, preferably water/ethanol, mixture preferably composed of60-80 parts alcohol and 40-20 parts water, and then evaporating off thewater/alcohol liquid, leaving a powdered extract residue referred toherein as “the hydroalcoholic extract”.

In yet another aspect, the weight ratio of the hydroalcoholic extract ofturmeric to the supercritical extract of turmeric is from about 8:1 toabout 12:1.

In an alternate aspect, the weight ratio of the supercritical extract ofrosemary to the hydroalcoholic extract of rosemary is from about 1.6:1to about 2.4:1.

In a still further aspect, the hydroalcoholic extract of gingercomprises from about 2.4% to about 3.6%, more preferably from about 2.7%to about 3.3%, and most preferably about 3.0%, by weight of pungentcompounds.

In another aspect, the supercritical extract of ginger comprises fromabout 24% to about 36%, more preferably from about 27% to about 33%, andmost preferably about 30%, by weight of pungent compounds; and fromabout 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%,and most preferably about 8%, by weight of zingiberene.

In a further aspect, the supercritical extract of turmeric comprisesfrom about 36% to about 54%, more preferably from about 40.5% to about49.5%, and most preferably about 45%, by weight of turmerones.

In another aspect, the supercritical extract of rosemary comprises fromabout 18.4% to about 27.6%, more preferably from about 20.7% to about25.3%, and most preferably about 23%, by weight of total phenolicantioxidants.

In yet another aspect, the supercritical extract of oregano comprisesgreater than about 4.0%, more preferably from about 4.5% to about 5.5%,and most preferably about 5.0%, by weight of total phenolicantioxidants.

In a still further aspect, the hydroalcoholic extract of turmericcomprises from about 5.6% to about 8.4%, more preferably from about 6.3%to about 7.7%, and most preferably about 7%, by weight of curcumin.

In another aspect, the hydroalcoholic extract of rosemary comprises fromabout 18.4% to about 27.6%, more preferably from about 20.7% to about25.3%, and most preferably about 23%, by weight of total phenolicantioxidants.

In a further embodiment, the hydroalcoholic extract of holy basilcomprises from about 1.6% to about 2.4%, more preferably from about 1.8%to about 2.2%, and most preferably about 2%, by weight of ursolic acid.

In a further aspect, the hydroalcoholic extract of green tea comprisesfrom about 36% to about 54%, more preferably from about 40.5% to about49.5%, and most preferably about 45%, by weight of polyphenols.

In another aspect, the hydroalcoholic extract of huzhang comprises fromabout 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%,and most preferably about 8%, by weight of reservatrols.

In another embodiment, the hydroalcoholic extract of Chinese goldthreadcomprises from about 4.8% to about 7.2%, more preferably from about 5.4%to about 6.6%, and most preferably about 6%, by weight of berberine.

In a further aspect, the hydroalcoholic extract of barberry comprisesfrom about 4.8% to about 7.2%, more preferably from about 5.4% to about6.6%, and most preferably about 6%, by weight of berberine.

In an alternate aspect, said composition comprises:

(A) from about 4.5% to about 7.5% by weight of the hydroalcoholicextract of ginger, wherein the extract comprises from about 2.4% toabout 3.6% by weight of pungent compounds;

(B) from about 5.5% to about 8.5% by weight of the supercritical extractof ginger, wherein the extract comprises from about 24% to about 36% byweight of pungent compounds and from about 6.4% to about 9.6% by weightof zingiberene;

(C) from about 1.0% to about 1.5% by weight of the supercritical extractof turmeric, wherein the extract comprises from about 36% to about 54%by weight of turmerones;

(D) from about 10.0% to about 16.0% by weight of the supercriticalextract of rosemary, wherein the extract comprises from about 18.4% toabout 27.6% by weight of total phenolic antioxidants;

(E) from about 4.0% to about 6.0% by weight of the supercritical extractof oregano, wherein the extract comprises greater than about 4.0% byweight of total phenolic antioxidants;

(F) from about 10.0% to about 16.0% by weight of the hydroalcoholicextract of turmeric, wherein the extract comprises from about 5.6% toabout 8.4% by weight of curcumin;

(G) from about 5.5% to about 8.0% by weight of the hydroalcoholicextract of rosemary, wherein the extract comprises from about 18.4% toabout 27.6% by weight of total phenolic antioxidants;

(H) from about 10.0% to about 16.0% by weight of the hydroalcoholicextract of holy basil, wherein the extract comprises from about 1.6% toabout 2.4% by weight of ursolic acid;

(I) from about 10.0% to about 16.0% by weight of the hydroalcoholicextract of green tea, wherein the extract comprises from about 36% toabout 54% by weight of polyphenols;

(J) from about 8.0% to about 12.0% by weight of the hydroalcoholicextract of huzhang, wherein the extract comprises from about 6.4% toabout 9.6% by weight of reservatrols;

(K) from about 4.0% to about 6.0% by weight of the hydroalcoholicextract of Chinese goldthread, wherein the extract from about 4.8% toabout 7.2% by weight of berberine;

(L) from about 4.0% to about 6.0% by weight of the hydroalcoholicextract of barberry, wherein the extract from about 4.8% to about 7.2%by weight of berberine; and

(M) from about 2.0% to about 3.0% by weight of the hydroalcoholicextract of Scutellaria baicalensis;

and wherein said composition further comprises:

(i) the supercritical extract of ginger and the hydroalcoholic extractof ginger at a weight ratio of from about 0.8 to about 1.4 parts ofsupercritical extract per 1 part of hydroalcoholic extract;

(ii) the hydroalcoholic extract of turmeric and the supercriticalextract of turmeric at a weight ratio of from about 8 to about 12 partsof hydroalcoholic extract per 1 part of supercritical extract; and

(iii) the supercritical extract of rosemary and the hydroalcoholicextract of rosemary at a weight ratio of from about 1.6 to about 2.4parts of supercritical extract per 1 part of hydroalcoholic extract.

In an alternate aspect, the composition comprises an additional agentselected from the group consisting of antineoplastic agents, growthinhibiting agents, and nutrients.

Set forth in Table 1 is a preferred embodiment of the orallyadministered composition, excluding inactive ingredients, as used in theinventive methods. The amounts recited in Table 1 represent thepreferred dosage of the ingredients listed.

TABLE 1 Herb Type Of Extract Plant Part Amount (mg) Rosemarysupercritical leaf 100 Rosemary hydroalcoholic (23% TPA 34.5 mg) leaf 50Turmeric supercritical (45% turmerones 4.5 mg) rhizome 10 Turmerichydroalcoholic (7% curcumin 7 mg) rhizome 100 Ginger supercriticalrhizome 54 (30% pungent compounds 16.2 mg 8% zingiberene) Gingerhydroalcoholic (3% pungent compounds) rhizome 46 Holy basilhydroalcoholic (2% ursolic acid 2 mg) leaf 100 Green tea hydroalcoholic(45% polyphenols 45 mg) leaf 100 Huzhang hydroalcoholic (8% reservatrols6.4 mg) root & rhizome 80 Chinese Goldthread hydroalcoholic (6%berberine) root 40 Barberry hydroalcoholic (6% berberine 2.4 mg) root 40Oregano supercritical (≧4.0% TPA 1.8 mg) leaf 40 Scutellaria Baicalensishydroalcoholic (5:1) root 20

Preferably, the composition set forth in Table 1 also includes extravirgin olive oil and yellow beeswax.

Methods of the Inventive Subject Matter

The compositions of the present inventive subject matter generallycomprise standardized supercritical CO₂ concentrated extracts from plantproducts (ginger, rosemary, tumeric root, holy basil, green tea,huzhang, Chinese goldthread, barberry, oregano, and Baikal skullcap)typically consumed in an Eastern diet.

The inventive compositions were investigated for theiranti-proliferative effects on human PC3 cells, and specifically analyzedfor their effects on eicosanoid metabolism in this prostate cancer cellline. The inventive compositions were observed to produce aconcentration-dependent inhibition of cloned COX-1, COX-2 and 5-LOXactivities, with inhibition of production of 5-HETE being greater thanthat of PGE2 formation.

Applied to intact PC3 cells, the inventive compositions were found to bemost potent against 12-LOX, followed by 5-LOX and then COX activities.Surprisingly, the appearance of 13-S-HODE in the PC3 cells was due tothe presence of this eicosanoid in the inventive composition itself, andnot to a stimulation of 15-LOX activity within the cells. Theconcentration dependent inhibition of proliferation of PC3 cells wasassociated with a selective G2/M arrest of the cell cycle and inductionof apoptosis as evidenced by flow cytometry staining of PC3 cells withannexin V and phosphatidyl inositol.

The inventive compositions also produced a concentration-dependentdown-regulation of 5-LOX and 12-LOX expression, although at highconcentrations an up-regulation of COX-2 expression was noted. Thephosphorylation status of several cell signal transduction proteins wasdetermined. The inventive compositions produced an increase in p21phosphorylation but down regulated phosphorylation of Rb and STAT1proteins. The decrease in pRb proteins was shown to be due to 12-LOXinhibition and a decline in 12-HETE levels in the cells. Addition of12-HETE back to the inventive compositions treated cells overcame theability of the inventive compositions to inhibit cell proliferation andconcordantly 12-HETE blocked the inventive compositions' ability todown-regulate phosphorylation of Rb protein.

It is concluded that the effective control of human prostate cancer cellproliferation with the inventive compositions is multi-mechanistic butin part involves regulation of aberrant tumor cell eicosanoid metabolismsuch as 12-LOX, application of plant-derived eicosanoid products such as13-(S)-HODE as well as restoration of Rb tumor suppressor proteinfunction through regulation of its phosphorylation status.

Further, other experiments demonstrated that while the compositions haveshown potent inhibition of cyclooxygenase (cloned COX-1 and COX-2)activities, the ability to reduce growth of human prostate tumor cellgrowth produced by this product is believed to be in large part due toCOX-2 independent mechanisms. Using a LC/MS/MS based method thatsimultaneously determines multiple eicosanoids in cells and tissues,changes in eicosanoid metabolism in cells and tissues produced by thisunique herbal composition were examined.

Endogenous levels of intracellular PGE2, 12-HETE as well as 5-HETEdeclined in a concentration-dependent manner upon exposure to thecompositions. In contrast, cellular levels of 15-HETE and 13-HODE wereelevated. The elevation of 13-HODE was dramatic but, as discussed above,was due to a high content of 13-HODE present in the herbal compositionitself and was not dependent on cellular generation of this importanteicosanoid. The 13-HODE levels were considered to be high enough toaccount for inhibition of tumor cell growth that was independentlyverified by addition of authentic 13-HODE to tumor cell cultures.

Exposure of human tumor cells (PC3 prostate cancer cells) to theinventive composition also resulted in the downregulation of 5-LOXexpression as determined by Western blot analyses. The ability of theinventive composition to block arachidonic acid mediated mouse earinflammation was also assessed. The inventive compositions producedsignificant inhibition of LTB4 synthesis and up-regulated the productionof 15-HETE.

Thus, the inventive subject matter relates to a method for modulating aneicosanoid metabolic process in cells of an animal in need thereof,which comprises administering to the animal an amount of a compositioneffective for regulating the activity of an eicosanoid oxygenase,

-   -   wherein the composition comprises therapeutically effective        amounts of supercritical extracts of rosemary, turmeric, oregano        and ginger; and therapeutically effective amounts of        hydroalcoholic extracts of holy basil, ginger, turmeric,        Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese        goldthread, and barberry.

In one aspect of the inventive subject matter, the cells are cancercells. In one embodiment, the cancer cells comprise prostate cancercells, breast cancer cells, lung cancer cells, colon cancer cells, or acombination thereof.

In an alternate embodiment, the eicosanoid metabolic process is aberrantmetabolism associated with cellular transformation to cancer, cancercell proliferation, cancer cell metastasis, cancer cell invasiveness,cancer cell-modulated angiogenesis, cancer cell-modulated apoptosissuppression, or a combination thereof.

Eicosanoid Metabolism. Arachidonic acid and its precursor, linoleicacid, along with linolinic acid, are present in animal fats and avariety of vegetable oils which are generally thought to be consumed ingreater quantities in the typical Western diet in comparison to Easterndiets. The elevated intake of these fatty acids provides increasedsubstrate availability for oxygenases such as lipoxygenases (LOX) andcyclooxygenases (COX), enzymes which are responsible for convertingeicosanoids such as arachidonic acid into signaling molecules such asprostaglandins, leukotrienes, lipoxins, 13-S-HODE, 5-HETE, and 12-HETE.In addition to their vital role as second messengers in many importantbiological pathways, these products of eicosanoid metabolism have beenimplicated in all aspects of tumor development, progression,proliferation, and metastasis.

Lipoxygenases and Eicosanoid Metabolism. Lipoxygenase enzymes (LOXs) areknown to be key regulators of cell survival and apoptosis in cells. Theyconstitute a heterogeneous family of lipid peroxidizing enzymes that arecategorized with respect to their regional specificity of arachidonicacid oxygenation. LOXs have been designated as 5-, 8-, 12-, and 15-LOXisoforms, which produce end products known as 5(S)-, 8(S)-, 12(S)-, and15(S)-HETEs. Application of LC/MS/MS analyses of COX and LOX metabolismin PC3 cells was undertaken using a previously published technique. Theresults showed that the inventive compositions were, as expected, aninhibitor of both COX-1 and COX-2 cloned enzyme activity. In addition,it also inhibited cloned 5-LOX activity and, in fact was more potent asa 5-LOX inhibitor than as a COX inhibitor. Treatment of PC3 cells inculture showed that the inventive composition's ability to inhibit PGE2formation through inhibition of COX was less than its ability to inhibit5-LOX and this, in turn was less than the inventive composition-mediatedinhibition of 12-LOX, an enzyme know to be important in proliferationand metastases of human prostate cancer.

12-HETE. Several lines of evidence now clearly implicate 12-LOX as aregulator of cancer cell development. The laboratory of K. Honn, inparticular, has contributed greatly to the understanding of the role ofplatelet type 12-LOX and its product 12(S)-HETE in human prostatecancer. They reported, for example, that 12-LOX is expressed in severalprostate cancer cells lines, that inhibitors of 12-LOX such as baicaleinor BHPP significantly inhibit prostate tumor metastases and that 12-LOXlevels are correlated with the grade and severity of human prostatetumors. A recent elegant report from this group has also shown thatbaicalein inhibition of prostate cell growth and proliferation isassociated specifically with 12-LOX inhibition and that the decline in12-HETE correlates with a loss of phosphorylated Rb proteins. Thedecreased phosphorylation of RB, in turn, results in RB proteinremaining bound to transcription factors required for DNA synthesis andthus results in inhibition of cancer cell proliferation.

There are many bioactive lipids involved in inflammation associated withcancers such as early stage prostate cancer. These include the welldocumented ability of PGE₂ to stimulate tumor cell proliferation. Thistypically occurs through an over-expression of COX-2 in tumors as wellthe more recent finding of a decrease in PGDH activity, the enzymeresponsible for metabolism of PGE₂. Lipoxygenase products such as 5-HETEfrom 5-lipoxygenase and 12-HETE from 12-lipoxygenase have also beenshown to specifically drive prostate tumor cell proliferation. Because12-HETE has been shown to be associated with prostate cancerproliferation, metastases and angiogenesis the suggestion thatinhibition of 12-LOX represents a potential therapeutic approach intreatment of prostate cancer has recently been made. Other eicosanoidssuch as 13-S-HODE, the product of 15-LOX-1 and 15-HETE, the product of15-LOX-2 appear to play opposing effects on signaling in cancers,especially prostate cancer, and may, in fact, be tumor type selective intheir effects.

To better understand the role of selected bioactive eicosanoids in theprogression of tissue inflammation towards malignant disease, Applicantshave undertaken examination of prostate cell related inflammationprofiles using an electrospray tandem mass spectrometry assay thatApplicants developed. This method permits determination of“mini-lipidomics” in cell or tissue samples through simultaneousanalyses of up to 10 cyclooxygenase, lipoxygenase and/or cytochrome P450derived eicosanoids at one time without the need for addition ofexogenous substrates such as arachidonic acid or linoleic acid.

To better understand the role of eicosanoids in human cancer Applicantsused this novel tissue inflammation assay in PC3 cells to determine theeffect of the inventive compositions on cyclooxygenase and lipoxygenasederived eicosanoids believed to be importance in proliferation of thishuman disease. Applicants have determined what effects, if any, theinventive compositions produced in eicosanoid metabolism beyondinhibition of cyclooxygenase activity. Our data suggest that thismultiherb product inhibits 5-LOX and 12-LOX activities. The laterfinding may be of special interest in that suppression of 12-LOXactivity was found to be associated specifically with inhibition oftumor cell proliferation and Rb phosphorylation, and thus return oftumor suppressor activity to hormone refractory PC3 prostate tumorcells.

Thus, in a preferred embodiment, the eicosanoid is selected from thegroup consisting of arachidonic acid and linolinic acid.

In a more preferred embodiment, the eicosanoid is arachidonic acid.

In a further aspect of the inventive subject matter, the eicosanoidoxygenase is cyclooxygenase-1, cyclooxygenase-2,5-lipoxygenase,12-lipoxygenase, or a combination thereof.

In a preferred embodiment, the eicosanoid oxygenase is 12-lipoxygenase.

In another preferred embodiment, the eicosanoid oxygenase is5-lipoxygenase.

Cyclooxygenases. The cyclooxygenase enzymes that mediate eicosanoidmetabolism are represented by two species, cyclooxygenase-1 (COX-1)which is constitutively expressed in many tissues and cyclooxygenase-2(COX-2) which is typically induced during disease states such asinflammation and cancer. Data from many molecular and cellular biologystudies have also suggested that the COX-2 gene is an early growthresponse gene affecting pathways that modulate apoptosis, proliferation,adhesion, angiogenesis and differentiation.

COX and especially COX-2 inhibition has been a main target foranti-inflammatory drug design for many years, however, the link betweenCOX-2 expression and cancer has only been more recently recognized.Observations from several population-based studies have documented asignificant decrease in the risk of colorectal cancer in people whoregularly take non-steroidal anti-inflammatory drugs that have potentCOX inhibitory activity. Histological studies that followed colorectaltumor development have determined that most human and animal colorectaltumors express elevated COX-2 levels, while adjacent normal colorectalepithelial cells have low to undetectable COX-2 levels. Similar to theseobservations, several laboratories have also reported that COX-2expression is elevated in prostate tumor cells during both initiationand progression compared to normal epithelial cells, however thisfinding is controversial.

It is clear though, that several pharmacological COX-2 inhibitory drugshave shown the ability to suppress prostate cancer cell growth in vitro,induce apoptosis, and suppress growth of human prostate tumor xenograftsin immunodeficient mouse models or transgenic models of prostate cancersuch as the TRAMP mouse. Given the controversy as to whether COX-2 is afactor in prostate cancer development or progression, several of theknown COX inhibitors are thought to have a variety of COX-independentanti-cancer effects and these actions appear to differ amongstinhibitors.

In this regard, it is very interesting that many plants which areprominent in regional diets contain substances that have COX inhibitoryactivity. Extracts from these plants, both in the crude form and asisolated components, have been found to have potent anti-inflammatoryand anti-cancer activities. Salicylic acid, for example, is atraditional inflammatory inhibitor agent found in willow tree bark andthe chemical derivative of this agent, aspirin, remains one of the mostcommonly used COX inhibitory substances in the world. In this study,Applicants considered the potential of a unique commercially availableherbal preparation, the inventive compositions, for their ability toaffect COX-1 and COX-2 enzyme activities and influence the behavior of acommonly used human prostate cancer cell model system, LNCaP cells. Asdiscussed in detail above, the inventive compositions are comprised often standardized herbal extracts, (rosemary, turmeric, ginger, holybasil, green tea, hu zhang, Chinese goldthread, barberry, oregano, andScutellaria baicalensis). Each of these herbs has been shown to containunique chemical constituents that influence COX activity or expression,and each has been studied for either anti-inflammatory or anti-canceractivity. This testing tends to focus on the predominant compound foundin any given herb, however there is reason to believe that there may beadditive benefit in the combination of multiple herbal/dietary agentsfor diseases such as cancer. The multiple and chemically diverseconstituents present in the inventive composition, each of which is anintegral component of the typical Asian diet, may be more effectiveagainst prostate cancer than any single herbal extract alone.

Thus, in an alternate aspect of the inventive subject matter, theeicosanoid oxygenase is cyclooxygenase-1, cyclooxygenase-2, or acombination thereof.

In a preferred embodiment, the eicosanoid oxygenase is cyclooxygenase-1.

In a further preferred embodiment, the eicosanoid oxygenase iscyclooxygenase-2.

In one alternative aspect of the inventive subject matter, theregulation of the activity of an eicosanoid oxygenase is oxygenaseinhibition.

In an alternate aspect of the inventive subject matter, modulation of aneicosanoid metabolic process comprises inhibiting NF-κB activity in thecells of the animal.

The inventive subject matter further relates to a method of delivering13-S-HODE to an animal in need thereof, comprising administering to theanimal a composition comprising therapeutically effective amounts ofsupercritical extracts of rosemary, turmeric, oregano and ginger; andtherapeutically effective amounts of hydroalcoholic extracts of holybasil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea,huzhang, Chinese goldthread, and barberry.

Additionally, the inventive subject matter relates to a method forinhibiting arachidonic acid-mediated inflammation in an animal in needthereof, comprising administering to the animal a composition comprisingtherapeutically effective amounts of supercritical extracts of rosemary,turmeric, oregano and ginger; and therapeutically effective amounts ofhydroalcoholic extracts of holy basil, ginger, turmeric, Scutellariabaicalensis, rosemary, green tea, huzhang, Chinese goldthread, andbarberry.

NF-κB. Nuclear factor KB is a family of Rel-domain containing proteinspresent in the cytoplasm of all cells, where they are kept in aninactive state by a family of anchorin-domain-containing proteins whichincludes IκBα, IκBβ, IκBγ, IκBε, bcl-3, p105 and p100. Under restingconditions, NF-κB consists of a heterotrimer of p50, p65, and IκBα inthe cytoplasm; only when activated and translocated to the nucleus isthe sequence of events leading to transcription initiated. Mostcarcinogens, inflammatory agents, and tumor promoters, includingcigarette smoke condensate, phorbol ester, okadaic acid, H₂O₂, and tumornecrosis factor (TNF), have been shown to activate an NF-κB activationpathway. The activation of NF-κB involves the phosphorylation,ubiquitination, and degradation of IκBα and phosphorylation of p65,which in turn leads to the translocation of NF-κB to the cell nucleus,where it binds to specific response elements in the DNA. Thephosphorylation of IκBα is catalyzed by IκBαkinase (IKK), which isessential for NF-κB activation by most agents. NF-κB has been shown toregulate the expression of a number of genes whose products are involvedin tumorigenesis. These include antiapoptotic genes (e.g. clap, suvivin,traf, cflip, bfl-1, bcl-2 and bc-xl), angiogenesis (cox-2, mmp-9, vegf),genes encoding adhesion molecules, chemokines, and inflammatorycytokines; and cell cycle regulatory genes (e.g., cyclin dl, c-myc).

Without being bound to a particular mechanism of action, Applicantsbelieve that the inventive compositions modulate the activity of thenuclear factor KB (“NF-κB”), which in turn regulates proliferation,invasion, and metastasis of tumor cells, inhibits feedback NF-κBactivation, and regulates expression of NF-κB-regulated gene products.In particular, Applicants have found that the inventive compositionsinhibit receptor activator of NF-κB ligand induced osteoclastogenesis,suppresses tumor necrosis factor (TNF)-induced invasion, and potentiatesthe apoptosis induced by TNF and chemotherapeutic agents. Further, theinventive compositions suppresses NF-κB activation induced by both TNFand cigarette smoke condensate. In addition, the inventive compositionsdownregulates the expression of NF-κB-regulated gene products involvedin anti-apoptosis, including inhibitor-of-apoptosis protein 1/2, Bcl-2,Bcl-xL, FADD like interleukin-1β converting enzyme (FLICE)/caspase-8inhibitory protein, TNF receptor-associated factor 1, and survivin, andfurther downregulates the expression of gene products involved inangiogenesis, including vascular endothelial growth factor,cyclooxygenase-2, intercellular adhesion molecule, and matrixmetalloproteinase. These results correlate with potentiation ofapoptosis induced by TNF and chemotherapeutic agents. OverallApplicants' results indicate that the inventive compositions suppressosteoclastogenesis, inhibits invasion, and potentiates apoptosis throughdownregulation of NF-κB activation and downregulation of NF-κB-regulatedgene products.

Applicants have determined some of the effects of the inventivecompositions on the NF-κB activation pathway and on the NF-κB-regulatedgene products which control tumor cell survival, proliferation,invasion, angiogenesis, and metastasis, finding that the inventivecompositions inhibits RANKL-induced osteoclastogenesis and TNF-inducedinvasion, and potentiates apoptosis induced by TNF and chemotherapeuticagents in various tumor cell lines. The expression of gene productsinvolved in anti-apoptosis, including IAP1, Bfl-1/A1, Bcl-2, TRAF1, andcFLIP, and expression of gene products involved in metastasis, includingMMP-9, COX-2, ICAM-1, and VEGF, were also downregulated by the inventivecompositions.

Applicants' data indicate that the inventive compositions suppress NF-κBactivated by TNF in human myeloid leukemia KBM-5 cells and cigarettesmoke condensate in lung adenocarcinoma H1299 cells. This is the firstinvestigation to examine the effect of the inventive compositions onNF-κB activated by different stimuli. These results suggest that theinventive compositions act at a step common to both these agents.Several genes that are involved in the proliferation and metastasis ofcancer have been shown to be regulated by NF-κB. (See, e.g., Aggarwal,B. B. (2004) Nuclear factor-kappa B: the enemy within. Cancer Cell, 6,203-208.) Applicants have shown that the inventive compositions inhibitthe expression of COX-2, MMP-9, and VEGF regulated by NF-κB.

Further, while recent reports have suggested that the inventivecompositions suppress both COX-1 and COX-2 enzymatic activities (See,e.g., Bemis, D. L., Capodice, J. L., Anastasiadis, A. G., Katz, A. E.and Buttyan, R. (2005) Zyflamend, a unique herbal preparation withnonselective COX inhibitory activity, induces apoptosis of prostatecancer cells that lack COX-2 expression. Nutr. Cancer., 52, 202-212),Applicants have shown that the inventive compositions inhibits theexpression of the COX-2 protein.

Applicants' data suggest that the inventive compositions exercise itsanti-cancer properties on NF-κB-regulated pathways through the directinhibition of NF-κB. NF-κB is known to regulate the expression of IAP1,xIAP, Bfl-1/A1, TRAF1, Bcl-2, cFLIP, and survivin, and theiroverexpression in numerous tumors has been linked to survival,chemoresistance, and radioresistance. (See, e.g., Takada, Y., Singh, S,and Aggarwal, B. B. (2004) Identification of a p65 peptide thatselectively inhibits NF-kappa B activation induced by variousinflammatory stimuli and its role in down-regulation of NF-kappaB-mediated gene expression and up-regulation of apoptosis. J. Biol.Chem., 279, 15096-15104.) Applicants' data indicate that the inventivecompositions treatment down-regulates most of these gene products.Previous reports have suggested that the inventive compositions induceapoptosis through a caspase-mediated pathway in human prostate cancercells (See, e.g., Bemis, et al., (2005)). Applicants' results also showthat the inventive compositions potentiate the apoptotic effects of TNF,taxol, and doxorubicin. These effects are similar to those that havebeen reported with a specific inhibitor of NF-κB (See, e.g., Takada, etal. (2004)).

Again without being bound to a particular mechanism of action,Applicants believe that antiproliferative, pro-apoptotic, anti-invasive,anti-osteoclastogenic, anti-angiogenic, and anti-metastatic effectsobserved upon administration of the inventive compositions are mediatedthrough suppression of NF-κB-regulated gene products. Whereas each ofthe herbs that are used in the formulation of the inventive compositionsare known to contain unique anti-inflammatory and anticancer compounds,one common property of the components of the inventive compositionsappears to be the ability to suppress NF-κB activation.

Considering the fact that the inventive compositions is derived fromnatural herbal sources and is readily available in health food andnutritional supplement source, make it a more convenient and desirablemeans for prevention and treatment of cancer than prescription cancerdrugs.

Thus, the inventive subject matter also relates to a method formodulating the level of NF-κB-regulated gene products in cells of ananimal in need thereof, comprising administering to the animal acomposition comprising therapeutically effective amounts ofsupercritical extracts of rosemary, turmeric, oregano and ginger; andtherapeutically effective amounts of hydroalcoholic extracts of holybasil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea,huzhang, Chinese goldthread, and barberry.

The animal in all of the inventive methods disclosed above may be amammal such as a mouse, rat, cat, dog, horse, cow, or other domesticatedanimal, or a human. In a preferred embodiment, the animal is human. Inaddition to uses for treating human diseases, disorders, and conditions,the inventive methods may have veterinary applications.

Routes of Administration

In a preferred embodiment, an orally administered composition is in theform of one or more capsules, one or more tablets, or one or more pills.

The inventive compositions are preferably delivered to the patient bymeans of a pharmaceutically acceptable carrier. Such carriers are wellknown in the art and generally will be in either solid or liquid form.Solid form pharmaceutical preparations which may be prepared accordingto the inventive subject matter include powders, tablets, dispersiblegranules, capsules, cachets and suppositories. In general, solid formpreparations will comprise from about 5% to about 90% by weight of theactive agent.

A solid carrier can be one or more substances which may also act asdiluents, flavoring agents, solubilizers, lubricants, suspending agents,binders or tablet disintegrating agents; it can also be encapsulatingmaterial. In powders, the carrier is a finely divided solid which is inadmixture with the viscous active compound. In tablets, the activecompound is mixed with a carrier having the necessary binding propertiesin suitable proportions and compacted to the shape and size desired.Suitable solid carriers include magnesium carbonate, magnesium stearate,talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoabutter, and the like. The term “preparation” is intended to include theformulation of the active compound with encapsulating materials as acarrier which may provide a capsule in which the active component (withor without other carriers) is surrounded by carrier, which is thus inassociation with it. Similarly, cachets are included. Tablets, powders,cachets, and capsules can be used as solid dosage forms suitable fororal administration. If desired for reasons of convenience or patientacceptance, pharmaceutical tablets prepared according to the inventivesubject matter may be provided in chewable form, using techniques wellknown in the art.

For preparing suppositories, a low melting wax such as a mixture offatty acid glycerides or cocoa butter is first melted, and the activeingredient is dispersed homogeneously therein as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions.As an example may be mentioned water or water/propylene glycol solutionsfor parenteral injection. Liquid preparations can also be formulated insolution in aqueous polyethylene glycol solution. Aqueous solutionssuitable for oral use can be prepared by dissolving the active componentin water and adding suitable colorants, flavors, stabilizers andthickening agents as desired. Aqueous suspensions suitable for oral usecan be made my dispersing the finely divided active component in waterwith a viscous material, i.e., natural Or synthetic gums, resins,methylcellulose, sodium carboxymethylcellulose, and other well knownsuspending agents. Liquid pharmaceutical preparations may comprise up to100% by weight of the subject active agent.

Also contemplated as suitable carriers are solid form preparations whichare intended to be converted, shortly before use, to liquid formpreparations for either oral or parenteral administration. Such liquidforms include solutions, suspensions, and emulsions. These particularsolid form preparations are most conveniently provided in unit dose formand as such are used to provide a single liquid dosage unit.Alternately, sufficient solid may be provided so that after conversionto liquid form, multiple individual liquid doses may be obtained bymeasuring predetermined volumes of the liquid form preparation as with asyringe, teaspoon, or other volumetric container. When multiple liquiddoses are so prepared, it is preferred to maintain the unused portion ofsaid liquid doses at low temperature (i.e., under refrigeration) inorder to retard possible decomposition. The solid form preparationsintended to be converted to liquid form may contain, in addition to theactive material, flavorants, colorants, stabilizers, buffers, artificialand natural sweeteners, dispersants, thickeners, solubilizing agents,and the like. The liquid utilized for preparing useful liquid formpreparations may be water, isotonic water, ethanol, glycerine, propyleneglycol, and the like as well as mixtures thereof. Naturally, the liquidutilized will be chosen with regard to the route of administration. Forexample, liquid preparations containing large amounts of ethanol are notsuitable for parenteral use.

The inventive pharmaceutical preparations may include one or morepreservatives well known in the art, such as benzoic acid, sorbic acid,methylparaben, propylparaben and ethylenediaminetetraacetic acid (EDTA).Preservatives are generally present in amounts up to about 1% andpreferably from about 0.05 to about 0.5% by weight of the pharmaceuticalcomposition.

Useful buffers for purposes of the inventive subject matter includecitric acid-sodium citrate, phosphoric acid-sodium phosphate, and aceticacid-sodium acetate in amounts up to about 1% and preferably from about0.05 to about 0.5% by weight of the pharmaceutical composition. Usefulsuspending agents or thickeners include cellulosics likemethylcellulose, carageenans like alginic acid and its derivatives,xanthan gums, gelatin, acacia, and microcrystalline cellulose in amountsup to about 20% and preferably from about 1% to about 15% by weight ofthe pharmaceutical composition.

Sweeteners which may be employed include those sweeteners, both naturaland artificial, well known in the art. Sweetening agents such asmonosaccharides, disaccharides and polysaccharides such as xylose,ribose, glucose, mannose, galactose, fructose, dextrose, sucrose,maltose, partially hydrolyzed starch or corn syrup solids and sugaralcohols such as sorbitol, xylitol, mannitol and mixtures thereof may beutilized in amounts from about 10% to about 60% and preferably fromabout 20% to about 50% by weight of the pharmaceutical composition.Water soluble artificial sweeteners such as saccharin and saccharinsalts such as sodium or calcium, cyclamate salts, acesulfame-K,aspartame and the like and mixtures thereof may be utilized in amountsfrom about 0.001% to about 5% by weight of the composition.

Flavorants which may be employed in the pharmaceutical products of theinventive subject matter include both natural and artificial flavors,and mints such as peppermint, menthol, vanilla, artificial vanilla,chocolate, artificial chocolate, cinnamon, various fruit flavors, bothindividually and mixed, in amounts from about 0.5% to about 5% by weightof the pharmaceutical composition.

Colorants useful in the inventive subject matter include pigments whichmay be incorporated in amounts of up to about 6% by weight of thecomposition. A preferred pigment, titanium dioxide, may be incorporatedin amounts up to about 1%. Also, the colorants may include other dyessuitable for food, drug and cosmetic applications, known as F.D.&C. dyesand the like. Such dyes are generally present in amounts up to about0.25% and preferably from about 0.05% to about 0.2% by weight of thepharmaceutical composition. A full recitation of all F.D.&C. and D.&C.dyes and their corresponding chemical structures may be found in theKirk-Othmer Encyclopedia of Chemical Technology, in Volume 5, at pages857-884, which text is accordingly incorporated herein by reference.

Useful solubilizers include alcohol, propylene glycol, polyethyleneglycol and the like and may be used to solubilize the flavors.Solubilizing agents are generally present in amounts up to about 10%;preferably from about 2% to about 5% by weight of the pharmaceuticalcomposition.

Lubricating agents which may be used when desired in the instantcompositions include silicone oils or fluids such as substituted andunsubstituted polysiloxanes, e.g., dimethyl polysiloxane, also known asdimethicone. Other well known lubricating agents may be employed.

The pharmaceutical preparation may also be prepared in a unit dosageform. In such form, the preparation is subdivided into unit dosescontaining appropriate quantities of the active component. The unitdosage form can be a packaged preparation, the package containingdiscrete quantities of preparation, for example, packeted tablets,capsules, and powders in vials or ampoules. The unit dosage form canalso be a capsule, cachet, or tablet itself or it can be the appropriatenumber of any of these in packaged form.

It is not expected that compounds of the inventive subject matter willdisplay significant adverse interactions with other synthetic ornaturally occurring substances. Thus, a compound of the inventivesubject matter may be administered in combination with other compoundsand compositions useful, for example, for treating cancer. In particularthe compounds of the inventive subject matter may be administered incombination with other compounds of the inventive subject matter,chemotherapeutic substances, and so forth.

The optimal pharmaceutical formulations will be determined by oneskilled in the art depending upon considerations such as the route ofadministration and desired dosage. See, for example, “Remington'sPharmaceutical Sciences”, 18th ed. (1990, Mack Publishing Co., Easton,Pa. 18042), pp. 1435-1712, the disclosure of which is herebyincorporated by reference. Such formulations may influence the physicalstate, stability, rate of in vivo release, and rate of in vivo clearanceof the present therapeutic agents of the inventive subject matter.

Dosage

Dosage levels on the order of about 0.001 mg to about 100 mg perkilogram body weight of the active ingredient compounds or compositionsare useful in the treatment of the above conditions, with preferredlevels ranging from 200 mg per day to 1600 mg per day. The compounds andcompositions of the inventive subject matter may usually be given in twoor three doses daily. Starting with a low dose (200-300 mg) twice dailyand slowly working up to higher doses if needed is a preferred strategy.The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration.

It is understood, however, that a specific dose level for any particularpatient will depend upon a variety of factors, including the activity ofthe specific compound employed; the age, body weight, general health,sex and diet of the patient; the time of administration; the rate ofexcretion; drug combination; the severity of the particular disorderbeing treated; and the form of administration. One of ordinary skill inthe art would appreciate the variability of such factors and would beable to establish specific dose levels using no more than routineexperimentation.

EXAMPLES

The following examples are illustrative of the inventive subject matterand are not intended to be limitations thereon. Unless otherwiseindicated, all percentages are based upon 100% by weight of the finalcomposition.

General Materials and Methods

Zyflamend®, obtained from New Chapter Inc. (St. Louis, Mo.), wasdissolved in dimethyl sulfoxide (“DMSO”) as a 10 mg/ml stock solutionand stored at −20° C. Bacteria-derived human tumor necrosis factor alpha(“TNF-α”), purified to homogeneity with a specific activity of 5×10⁷U/mg, was kindly provided by Genentech, Inc. (South San Francisco,Calif.). Penicillin, streptomycin, RPMI 1640 medium, Iscove's modifieddulbecco medium (“IMDM”), D-MEM/F12 medium and fetal bovine serum(“FBS”) were obtained from Invitrogen (Grand Island, N.Y.). Thefollowing polyclonal antibodies were obtained from Santa CruzBiotechnology, Inc. (Santa Cruz, Calif.): anti-matrix metalloproteinase9 (“MMP-9”); anti-intercellular adhesion molecule (“ICAM”);antiinhibitor-of-apoptosis protein 1/2 (“IAP 1/2”); anti-Bcl-2;anti-Bfl-1/A1; and anti-TNF receptor-associated factor (“TRAF1”).Anti-COX-2 and XIAP were obtained from BD Biosciences (San Diego,Calif.). Cigarette smoke condensate (“CSC”), prepared as described inAnto, R. J., Mukhopadhyay, A., Shishodia, S., Gairola, C. G. andAggarwal, B. B. (2002) Cigarette smoke condensate activates nucleartranscription factor-kappa B through phosphorylation and degradation ofI kappa B(alpha): correlation with induction of cyclooxygenase-2.Carcinogenesis, 23, 1511-1548, was kindly supplied by Dr. G. Gairola(University of Kentucky, Lexington, Ky.). An anti-vascular endothelialgrowth factor (“VEGF”) was purchased from NeoMarkers (Fremont, Calif.).Survivin antibody was obtained from R&B Systems (Minneapolis, Minn.). AnFADD-like interleukin-1β-converting enzyme(“FLICE”)/caspase-8-inhibitory protein (“cFLIP”) antibodies were kindlyprovided by Imgenex (San Diego, Calif.).

The cell lines used in Applicants' work include human non-small celllung carcinoma (H1299), human myelogenous leukemia (KBM-5), and humanmultiple myeloma (U266), mouse macrophages (RAW 264.7) cell lines wereobtained from the American Type Culture Collection (Manassas, Va.).H1299, and U266 cells were cultured in RPMI 1640 medium with 10% FBS.KBM-5 cells were cultured in IMDM with 15% FBS. RAW 264.7 cells werecultured in D-MEM/F12 medium supplemented with 10% FBS. All the mediawere supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Example 1 Inhibition of Cell Growth

As a general starting point, the relative ability of a product or drugto inhibit cell growth using established rodent and human cell lines inculture is investigated. The purpose of this is simply to be able todiscern those concentrations that are either ineffective or toxic fromthose that are significantly inhibitory or “effective”. Any explorationof mechanisms of action of a given product should be done atconcentrations that produce some but not profound inhibition of growthor massive cell death. In addition, these kinds of studies provide aninitial estimate of time-dependent changes in cell events. That is, whendrug-mediated events occur relative to the first indications of growthinhibition or cell death is discernable. This, in turn, may suggest asequence of events that are involved.

Most of the studies focused upon include using human prostate cancercell lines. The reason for this includes the fact that this is a cellline of interest, the cell lines have been characterized by us foreicosanoid metabolism and Applicants are involved in an ongoing clinicaltrial of the inventive compositions in the treatment/prevention of PIN.However, as the data are obtained, use of other cell lines and animalmodels that have little to do with prostate cancer itself but are,instead, useful for understanding general anti-inflammatory activity andmechanisms might be used.

FIG. 1 shows the inhibitory effect of the inventive compounds on growthof human A549 lung cancer and human PC3 prostate cancer cell lines. Theoral formulation of the composition was used. The composition was addedto cells in culture and relative inhibition of cell growth was assessedafter 72 hours of continuous exposure to drug. The “MTT” method was usedto assess cell proliferation versus untreated control cell populations.The greater inhibition of the growth of prostate PC3 cells compared tothat of lung adenocarcinoma cells is apparent.

Likewise, FIG. 8 and Table 2 depict the inhibitory effect of thecompound on growth of human breast cancer MCF7 and MDA231 cell lines.Again, the oral formulation of the inventive compositions was used. Thecomposition was added to cells in culture and relative inhibition ofcell growth was assessed after 72 hours of continuous exposure to drug.The “MTT” method was used to assess cell proliferation versus untreatedcontrol cell populations. The MCF7 cells were more sensitive to thecomposition exposure than were the MDA231 cells.

TABLE 2 Cell Line *IC₅₀ PC3 human prostate 146 Epi human normalepithelial >1000 MCF7 human breast cancer 27 (estrogen sensitive) MDA231human breast cancer 126 (estrogen refractory) A549 human lungadenocarcinoma 435

-   -   *The IC₅₀ is defined as that concentration (ug/ml) of material        that produces inhibition of growth of cells by 50% (relative to        untreated cells) under defined conditions (typically duration)        of exposure to that agent.

The relative lack of cytotoxicity against the only “normal” humanepithelial cell line tested is clearly of interest for purposes ofpatient tolerance of the medication and compliance with theadministration protocol. The sensitivity of human breast MCF-7 estrogensensitive cell line to the composition is of special interest, and thiscell line is definitely the most sensitive one tested to date.Applicants are unaware of any compounds in the plant extracts used tomake up the inventive composition which block estrogen binding, whichApplicants believe rule out this alternate explanation for the observedhigh sensitivity.

Example 2 Ability of the Inventive Composition To Inhibit Cyclooxygenase(COX) and Lipoxygenase (LOX) Enzymes

Applicants have determined the relative effect of the inventivecompositions against COX-1, COX-2, and other eicosanoids of interest.These studies have indicated that the composition inhibits both COX-1and COX-2 (as cloned enzymes), as Applicants expected based upon thevaried anti-inflammatory components obtained from the different herbalextracts that comprise the inventive composition. The data also indicatethat the composition is a useful inhibitor of 5-lipoxygenase (5-LOX) aswell. No selective change in eicosanoid products derived from 15-LOX-2was observed. The data also demonstrate that the compositions inhibit12-lipoxygenase in a concentration-dependent manner. The data in FIGS. 2and 3 indicate that the composition is potent in terms of producinginhibition of cloned COX enzymes as well as enzymes in cells in culture.Only small microliter amounts of the inventive composition in liquidwere sufficient to produce a significant inhibition of cyclooxygenaseenzymes. This is especially impressive when it is recalled that 60% ofthis particular formulation of the composition is olive oil, whichitself has no effect on eicosanoid metabolism.

FIG. 2 depicts the effect of the composition on formation of PGE2 usingcloned COX-1 (ovine) and COX-2 (human) enzymes was measured byincubation of 10 uM AA with the enzymes (15 IU) in 0.1 M Tris-HClbuffer, pH 8.0, containing 5 mM EDTA, 2 mM phenol, and 1 uM hematin.Aliquots of the composition were added to tubes prior to addition of AA.Control incubations did not contain the composition. The bars with theasterisk represent the “olive oil” control. Incubations were carried outat 37° C. for 15 min. Reactions were stopped by addition of 1 N citricacid. Eicosanoids were then extracted using a hexane:ethyl acetate (1:1)solvent mixture; the extract was brought to dryness under nitrogen. PGE2formed during the incubation was extracted as previously described andthen analyzed using our published LC/MS/MS method (Yang et al. AnalBiochem. 308: 168-177, 2002). The data depicted in FIG. 2 suggest thatthe inventive composition inhibits the formation of PGE2 by both COX-1and COX-2, although the product is more potent at inhibiting COX-1 thanCOX-2.

FIG. 3 shows the effect of the composition on formation of PGE2 and5-HETE in PC3 cells. Varying concentrations of the composition wereadded to cell cultures in fresh serum-free medium supplemented with 15uM BSA and incubated at 37° C. for min. Arachidonic acid (100 uM) andcofactors were then added. After 10 min cells were washed and extractedto determine eicosanoid formed within the cells (see legend to FIG. 2).The data indicate a concentration-dependent inhibition of both PGE2 aswell as 5-HETE in PC-3 cells. Data are presented as Mean±SD (n=3). *Indicates P<0.05 relative to controls.

Example 3 Effect of the Inventive Compositions on Cellular Expression of5-Lipoxygenase

An anti-inflammatory agent can achieve its pharmacologic effect throughmany different mechanisms. A given product can inhibit selective enzymesinvolved with formation of inflammation related molecules, for exampleinhibition of COX-2 to decrease formation of PGE2 or inhibition of5-lipoxygenase to decrease formation of 5-HETE. The compositions alsoinhibit activation transcription factors that are known to be directlyinvolved in activation of inflammation related genes, such ascurcumin-mediated inhibition of activation of NF-kB.

In addition, a given product may also act to decrease the actualsynthesis, and therefore expression, of the enzyme in a cell or tissue,rather than directly inhibit it. The initial data we have obtainedindicates that the inventive compositions provide aconcentration-dependent inhibition of the relative expression of5-lipoxygenase in cells in addition to its inhibition of enzymeactivity. The relative inhibition of enzyme expression is not a generalphenomenon, as the composition does not appear to inhibit the relativetissue expression of COX-2.

Example 4

Identification of 13-S-HODE in the Inventive Composition

FIG. 4 shows the Concentration-dependent effect of the inventivecomposition on eicosanoid metabolism in human A549 lung cancer cells,showing apparent concentration-dependent increase in formation of13-S-HODE. Cells (1×10⁶) were allowed to attach overnight to the tissueculture plate and were then treated for 24 hours with differentconcentrations of the composition as shown in the Figure. Cells werethen harvested and extracted for eicosanoid analyses by LC/MS/MS. Thedata indicate a small inhibition (32%) of PGE2 at a compositionconcentration of 2 ul/ml but also show an “apparent” dramatic formationof 13-S-HODE at 1 and 2 ul/ml.

The effect of the composition on conversion of arachidonic acid tovarious eicosanoid products in several cell lines was examined. The datashown above suggest that, at least for human lung A459 cells, thecomposition dose not have a large effect on cyclooxygenase orlipoxygenase enzymes. The concentration-dependent increase in 13-S-HODE,the product of 15-LOX-1 enzyme is, however, striking. Such an increasein 13-S-HODE can arise from either an induction of the enzyme itself, orenzyme activity, addition of a suitable substrate such as linoleic acidthat would preferentially give rise to 13-S-HODE, or through anothermechanism. Induction of 15-LOX-1 does, in fact, happen when NSAIDs areadded to colon cancer cells and this serves as a partial explanation forthe beneficial effect of aspirin as a preventive agent in colon cancer.However, by Western blot, no significant induction of 15-LOX-1 enzyme asa result of incubation with the composition was observed. The inventivecomposition was also examined for relative content of linoleic acid, asubstrate that can give rise to 13-S-HODE through 15-LOX-1 enzyme. Usinga GC/MS/MS instrument, only a minimal amount of linoleic acid was found.Examination of the composition itself, however, indicated the presenceof a high amount of 13-S-HODE as discussed in Example 6 below.

FIG. 5 shows mass spectra data showing presence of 13-S-HODE in theinventive compositions. FIG. 5 represents three plots of relatedinformation. The uppermost plot is a total ion chromatogram ofdeuterated 13-S-HODE (designated as 13-S-HODE-d4). The four deuteriumatoms provide a higher molecular weight (addition of 4) to the massweight of 13-S-HODE itself. The mass weight of the deuterated product(indicated by the mass to charge ratio on the upper right is “299”. Thisis basically the mass weight of this product. The notation of 299>281reflects the fact that Applicants have the instrument select for themass weight of 299 and then collide the molecule with argon (an inertgas) to produce a characteristic “daughter” ion at 281. This daughterion can be used for quantitative purposes.

The middle plot indicates the ion trace for authentic 13-S-HODE (25ng/ml) purchased from Cayman Chemicals (Ann Arbor, Mich.). It has a massweight of 295.3 and this is indicated in the plot. Applicants look atthe daughter ion at 277.2 to characterize the compound. That is, thechances of any one given molecule having a mass weight of 277.2 thatalso “breaks” to produce a daughter ion of 277.2 is almost nil exceptfor the actual molecule of interest: 13-S-HODE.

The bottom plot is an extract of the inventive composition. The massspec was instructed to look for all molecules with a mass weight of295.3. Applicants then set the instrument to look at only characteristicdaughter ions at 277.2. The fact that the instrument found them . . . inabundance . . . indicates beyond a shadow of a doubt that 13-S-HODE ispresent in the inventive compositions. The extract was obtained fromonly 5 ul of the composition. The two numbers by each peak represent theretention time of the peak (e.g. 6.69 min) and the relative ionabundance. The later is analogous to the “amount” of product within thepeak. The fact that an authentic standard of 13-S-HODE at 25 ng/mlprovided a peak amount of 864,412 while only 5 ul of the compositionproduces a peak “amount” of 6,338,606 clearly indicates the high amountof this eicosanoid within the composition. What is important to know isthat this is “take it to the bank” proof that the composition contains alot of 13-S-HODE.

To assure that 13-S-HODE is the substance being dealt with, the presenceof 13-S-HODE in the inventive composition was assayed using an antibodybased Enzyme Immunoassay kit. The antibody provides the specificity fordetecting 13-S-HODE. The EIA analyses confirmed the presence of a highamount of 13-S-HODE in the inventive compositions.

An anti-inflammatory agent can achieve its pharmacologic effect throughmany different mechanisms. For example, a given product can inhibitselective enzymes involved with formation of inflammation relatedmolecules (e.g. inhibition of COX-2 to decrease formation of PGE₂ orinhibition of 5-lipoxygenase to decrease formation of 5-HETE). Theproduct could also inhibit activation transcription factors that areknown to be directly involved in activation of inflammation relatedgenes (e.g. curcumin mediated inhibition of activation of NF-kB). Inaddition, a given product may also act to decrease the actual synthesisand therefore expression of the enzyme in a cell or tissue rather thandirectly inhibit it. The initial data obtained indicates that theinventive composition can, in fact, lead to a concentration-dependentinhibition of the relative expression of 5-lipoxygenase in cells inaddition to inhibition of enzyme activity. The relative inhibition ofenzyme expression is not a general phenomenon as the composition doesnot appear to inhibit the relative tissue expression of 12-LOX. Nor isthe disappearance (down-regulation) of expression of 5-LOX a generalresult of exposure to 5-LOX inhibitors to cells. Although not in thisreport, the relative effect of 5-LOX protein content (Western blot)after PC3 cells had been exposed to the established 5-LOX inhibitorZileuton was examined. As shown in FIG. 9, while Zileuton is a potent5-LOX inhibitor (and is useful for treatment of asthma because of this),it did not produce any change in relative 5-LOX enzyme content withincells.

One of the interesting things is the concentration dependent increase inCOX-2 protein expression that occurs as a result of exposure of cells tothe composition. At first glance this would appear to be exactly whatApplicants would NOT want to happen. After all, inhibition of COX-2 isthe foundation for a large pharmaceutical business. But, littlerecognized is the fact that Celebrex (celecoxib) is a known inducer ofCOX-2! Inflammation stays in check because enzyme activity is inhibitedwhile Mother nature tries her best to overcome that with increasedsynthesis of the enzyme. In other words, people on COX-2 inhibitors dojust fine . . . until they stop taking them.

Example 5 In Vivo Determination of Anti-Inflammatory Effect of theInventive Compositions Mouse Ear Model

A very simple yet effective animal model of inflammation has been usedfor the past several years. The right ear of the test mouse ispretreated with a presumed anti-inflammatory agent then left alone for30 minutes. An inflammatory agent (typically arachidonic acid (AA) inacetone) is then applied to that same ear. The vehicle is applied to theleft ear as a control. A fixed period of time after application of AA,the mice is anesthetized and then a round ear punch is used to obtain auniform segment of tissue. The difference in weight of tissue from eachmouse ear punch serves as a relative index of the relative prevention ofinflammation. After weighing, the ear punch sample is then rapidlyfrozen for subsequent analyses of specific eicosanoid metabolism throughuse of our LC/MS/MS method.

FIG. 6 shows the anti-inflammatory effect of the inventive compositionon mouse ear edema. The composition (10 ul) was administered topicallyto the right ear and only the acetonevehicle was applied to the leftear. One hour following AA treatment, the mouse was anesthetized and theears “punched” to obtain a uniform piece of tissue. The weight of theright ear is then subtracted from that of the left ear to measure edema.The data reflect the relative ability of a given test substance toinhibit AA-mediated ear edema. Data are presented as mean+/−SD from 10animals. * P<0.001 treatment versus AA (edema) control (AA mediatedinflammation).

The data in FIG. 6 shows that the composition produces significantinhibition of arachidonic acid mediated inflammation.

Likewise, FIG. 10 shows the effect of the inventive composition onarachidonic acid mediated mouse ear edema. Treatment groups consistedof 1) olive oil control, 2) acetone control, 3) olive oil+AA, 4) Theinventive composition (10 ul)+AA, 5) The inventive composition (5ul)+AA, and 6) The inventive composition (2.5 ul)+AA. Data are providedas Mean±SD (n=10 mice/group). Once again, the composition inhibited theedema produced by AA. As shown in FIG. 10, a maximal inhibition of edemawas obtained at all three composition “doses” although it appeared thatthe use of 2.5 ul of the composition may have been slightly lesseffective than either of the two higher concentrations.

Example 6 The Herbal Source and Relative Importance of 13-S-HODE in theInventive Compositions

In analyzing the relative eicosanoid content of the liquid form of thecomposition, it was surprising to see the large amount of 13-S-HODE.These analyses were done using LC/MS/MS in which an authentic deuteratedstandard was used to make sure we had the correct peak identity. To makedoubly sure of this observation, the liquid composition was alsoanalyzed using an enzyme linked immunoassay kit especially prepared fordetermination of 13-S-HODE. The results from the EIA analysis confirmedthe mass spectrometry data and are shown in Table 3.

TABLE 3 Relative content of eicosanoids contained within the inventivecomposition PGE2 15-HETE 12-HETE 13-S-HODE Eicosanoid 0.10* 7.25 1.3547.59 *Data are provided as ng eicosanoid/5 ul of oral composition

The fact that the oral form of the composition is 60% olive oil (whichdoes not contain 13-S-HODE) indicates that the relative content of13-S-HODE in extract material is even higher than is shown above.

FIG. 7 shows the effect of 13-S-HODE on proliferation of various cancercell lines. The data above indicate that 13-S-HODE, the product of theenzyme 15-LOX-2, can totally inhibit the growth of human tumor prostate(PC3) and colon (LS174) but not lung adenocarcinoma (A549) cell growth.The relative ability of 13-S-HODE to inhibit prostate and colon cancercell lines with an IC₅₀ of about 3-4 uM. This is of importance whencompared to the content of 13-S-HODE in the inventive composition. Thatis, as shown in Table 2 above there is sufficient 13-S-HODE within thecomposition to account for inhibition of cell growth. No doubt otherfactors within the composition also contribute to its ability to inhibitprostate and colon tumor cell growth but the ability of 13-S-HODE withinthe composition to be able to do this is of obvious interest.

The relative eicosanoid content of the liquid form of the compositionwas examined and it was surprising to see the large amount of 13-S-HODE.These analyses were done using LC/MS/MS in which an authentic deuteratedstandard was used to make sure the correct peak was identified. To makedoubly sure of the observation, the liquid composition was also analyzedusing an enzyme linked immunoassay kit especially prepared fordetermination of 13-S-HODE. The results from the EIA analysis confirmedthe mass spectrometry data. The fact that the oral form of thecomposition is 60% olive oil (which does not contain 13-S-HODE)indicates that the relative content of 13-S-HODE in extract material iseven higher than is shown above.

A search for the source of the eicosanoid product 13-S-HODE amongst thedifferent herb and plant extract components that are used to make thecomposition was undertaken. The approach is to either solubilize theextract or subject the extract to a organic solvent mixture that will,in turn, pull out lipid soluble eicosanoids. These are then dried undernitrogen and reconstituted in a fixed volume of buffer compatible withthe mass spectrometry equipment. Another aliquot is used for analyses ofrelative linoleic acid content using a GC/MS/MS. The rationale behindthis is that 13-S-HODE can either be present as such in the extracts orit could also be derived through an enzymatic reaction in cells inculture if the cells contained 15-LOX-1 and were supplied with thesubstrate linoleic acid.

The data in Table 4 below show quite convincingly that, although thereare extracts that contain both linoleic acid and 13-S-HODE, theeicosanoid is itself a component of several of the herbal extracts thatare used to make the inventive composition. The fact that 13-S-HODE isabsent from some extracts (e.g. green tea) is also interesting and lendscredence to the fact that are measurements are correct.

TABLE 4 13-HODE content Linoleic acid content Composition component(ng/mg extract) (ug/mg extract) Tumeric PSE 32.55 0.78 Rosemary SCE 0.081.86 Holy Basil 1.42 1.15 Green tea 0.12 None detected Huzhang 0.14 1.50Ginger SCE 62.79 33.79 Oregano SCE 2.69 27.32 Barberry 10.41 0.27Rosemary 0.36 1.46 Tumeric SCE 16.30 31.10 Chinese Goldenthread 13.460.81 Scutellaria 0.64 None detected Ginger PSE 17.20 0.37

Both the determinations of 13-HODE as well as linoleic acid have beenperformed three times with comparable results. The 13-S-HODE wasdetermined using LC/MS/MS while linoleic acid content was measured usinga GC/MS/MS. The sources of the composition's 13-S-HODE appear to bemostly due to ginger and tumeric. It is interesting that this particulareicosanoid is present in both the PSE and the SCE extracts of ginger andto a lesser extent in tumeric root as well Notably, the presence of highamounts of 13-HODE is not always associated with high amounts oflinoleic acid, the substrate for 15-LOX-1 which produces 13-S-HODE.

Example 7 Osteoclast Differentiation Assay

To determine the effect of the inventive compositions on receptoractivator of NF-κB ligand (RANKL)-induced osteoclastogenesis, Applicantscultured RAW 264.7 cells, which can differentiate into osteoclasts byRANKL in vitro. RAW 264.7 cells were cultured in 24-well dishes at adensity of 1×10⁴ cells per well and allowed to adhere overnight. Themedium was then replaced, and the cells were coincubated with differentconcentrations of the inventive compositions and 5 nM RANKL. At days 5,the cells were stained for tartrate-resistant acid phosphatase (TRAP)expression, as previously described using an acid phosphatase kit(Sigma-Aldrich), and the TRAP-positive multinucleated osteoclasts (>3nuclei) per well were counted.

RAW 264.7 cells (1×10⁴ cells/well) were incubated either alone or in thepresence of 5 nM RANKL with 0.8 mg/ml the inventive compositions for 5days and stained for TRAP expression. As shown in FIG. 11(A),TRAP-positive cells were photographed (original magnification, 100×).

RAW 264.7 cells (1×10⁴ cells/well) were incubated either alone or in thepresence of 5 nM RANKL with The inventive compositions at the indicatedconcentrations for 5 days and stained for TRAP expression.Multinucleated (three nuclei) osteoclasts were counted, with the resultsdepicted graphically in FIG. 11(B).

It is shown that the inventive compositions suppress RANKL-inducedosteoclastogenesis. Because RANKL, a member of the TNF superfamily,induces osteoclastogenesis through the activation of NF-κB, Applicantsdetermined whether the inventive compositions can suppress RANKL-inducedosteoclastogenesis. Applicants found that RANKL induced osteoclastdifferentiation, as indicated by the expression of TRAP, as shown inFIG. 11(A), and that the inventive compositions suppressed it in adose-dependent manner, as shown in FIG. 11(B).

Example 8 Invasion Assay

The membrane invasion culture system was used to assess cell invasionbecause invasion through the extracellular matrix is a crucial step intumor metastasis. The BD BioCoat Tumor Invasion system is a chamber thathas a light-tight polyethelyene terephthalate membrane with 8μm-diameter pores and is coated with a reconstituted basement membranegel (BD Biosciences, San Diego, Calif.). A total of H1299 (2.5×10⁴cells) were suspended in serum-free medium and seeded into the upperwells. After incubation overnight, cells were coincubated with differentconcentrations of the inventive compositions and 1 nM TNF for a further24 h in the presence of 1% FBS. The cells that invaded through theMatrigel (i.e., those that migrated to the lower chamber duringincubation) were stained with 4 μg/ml Calcein AM (Molecular Probes,Eugene, Oreg.) in PBS for 30 min at 37° C. and scanned for fluorescencewith a Victor3 multi-plate reader (Perkin Elmer Life and AnalyticalSciences, Boston, Mass.); fluorescent cells were counted.

H1299 cells (2.5×10⁴ cells/well) were seeded into the upper wells of aMatrigel invasion chamber overnight in the absence of serum, coincubatedwith 0.3 mg/ml the inventive compositions and 1 nM TNF for 24 h in thepresence of 1% serum, and then subjected to invasion assay, with theresults depicted graphically as shown in FIG. 12. The value for none ofthe inventive compositions and no TNF was set to 1.0.

It is shown that the inventive compositions suppresses TNF-induced tumorcell invasion activity. It is known that NF-κB regulates the expressionof gene products (e.g., MMP-9, COX-2, and VEGF) that mediate tumor cellinvasion. Whether the inventive compositions can modulate TNF-inducedtumor cell invasion activity, was investigated in vitro. To determinethis, tumor cells were seeded to the top chamber of the matrigelinvasion chamber with TNF in the presence or absence of the inventivecompositions, and then examined for invasion. As shown in FIG. 12,TNF-induced tumor cell invasion by almost 2 fold and the inventivecompositions suppressed this activity in a dose-dependent manner. Theinventive compositions alone had no effect on invasion activity.

Example 9 LIVE/DEAD Assay

To measure apoptosis, Applicants used the LIVE/DEAD assay (MolecularProbes, Eugene, Oreg.), which determines intracellular esterase activityand plasma membrane integrity. This assay employs calcein, a polyanionicdye, which is retained within the live cells and provides greenfluorescence. It also employs the ethidium monomer dye (redfluorescence), which can enter the cells only through damaged membranesand bind to nucleic acids but is excluded by the intact plasma membraneof live cells. Briefly, 1×10⁶ cells were incubated with 0.5 mg/ml of theinventive compositions for 24 h and then treated with 1 nM TNF orvarious chemotherapeutic agents for 16 h at 37° C. Cells were stainedwith the LIVE/DEAD reagent (5 μM ethidium homodimer, 5 μM calcein-AM)and then incubated at 37° C. for 30 min. Cells were analyzed under afluorescence microscope (Labophot 2; Nikon, Tokyo, Japan).

Human multiple myeloma U266 cells (1×10⁶ cells/ml) were serum starvedfor 24 h and then incubated with 1 nM TNF, 1 nM taxol and 300 nMdoxorubicin alone or in combination with (0.5 mg/ml) of the inventivecompositions as indicated for 24 h. Cell death was determined by calceinAM based LIVE/DEAD assay, as shown in FIG. 13. Red color highlights deadcells, and green color highlights live cells.

The inventive compositions potentiate the apoptotic effects of TNF andchemotherapeutic drugs. Because NF-κB activation has been shown toinhibit the apoptosis induced by various agents, whether the inventivecompositions will modulate the apoptosis induced by TNF andchemotherapeutic agents, was investigated. The effect of the inventivecompositions on TNF and chemotherapeutic agent-induced apoptosis wasexamined by the LIVE/DEAD assay. The LIVE/DEAD assay, which measuresintracellular esterase activity and plasma membrane integrity, indicatedthat the inventive compositions enhances the apoptotic effects of TNF,taxol, and doxorubicin against tumor cells.

Example 10 Electrophoretic Mobility Shift Assays of NF-κB Activation

To determine NF-κB activation, Applicants carried out electrophoreticmobility shift assays (EMSA) essentially as previously described inChaturvedi, M. M., Mukhopadhyay, A. and Aggarwal, B. B. (2000) Assay forredox-sensitive transcription factor. Methods Enzymol., 319, 585-602.Briefly, nuclear extracts (1×10⁶ cells/ml) were incubated with32P-end-labeled 45-mer double-stranded NF-κB oligonucleotide (15 μg ofprotein with 16 fmol of DNA) from the human immunodeficiency virus longterminal repeat, 5′-TTGTTACAA GGGACTTTC CGCTG GGGACTTTC CAGGGAGGCGTGG-3′(boldface indicates NF-κB-binding sites), for 30 min at 37° C., and theDNA-protein complex formed was separated from free oligonucleotide on6.6% native polyacrylamide gels. A double stranded mutatedoligonucleotide, 5′-TTGTTACAA CTCACTTTC CGCTG CTCACTTTCCAGGGAGGCGTGG-3′, was used to examine the specificity of binding ofNF-κB to the DNA. The dried gels were visualized, and the radioactivebands were quantitated using a Storm 820 phosphorimager with theImageQuant software program (Amersham, Piscataway, N.J.).

KBM-5 cells (2×10⁶ cells/ml) were preincubated with indicatedconcentrations of the inventive compositions for 1 h, treated with 0.1nM TNF for 30 min. The nuclear extracts were assayed for NF-κBactivation by EMSA, as shown in FIG. 14(A).

The inventive compositions suppress NF-κB activation in a dose- andtime-dependent manner. As NF-κB plays an important role in apoptosis andin cell invasion, Applicants examined the effect of the inventivecompositions on activation of this transcription factor. Applicantsfirst investigated the effect of the inventive compositions on theactivation of NF-κB induced by TNF in human myelogenous leukemia (KBM-5)cells. DNA-binding assay (EMSA) results showed that the inventivecompositions alone had no effect on NF-κB activation. However, itinhibits TNF-mediated NF-κB activation in a dose-dependent manner, asshown in FIG. 14(A).

KBM-5 cells (2×10⁶ cells/ml) were preincubated with 1 mg/ml of theinventive compositions for the indicated times and then treated with 0.1nM TNF for 30 min. The nuclear extracts were assayed for NF-κBactivation by EMSA, as shown in FIG. 14(B). The suppression of NF-κBactivation by The inventive compositions were also found to betime-dependent.

The inventive compositions inhibit cigarette smoke-induced NF-κBactivation. H1299 cells were preincubated with the inventivecompositions (1 mg/ml) for 1 h, then treated with cigarette smoke (10μg/ml) for 1 h. The nuclear extracts were assayed for NF-κB activationby EMSA, as shown in FIG. 14(C).

The inventive compositions block NF-κB activation induced by cigarettesmoke condensate. Applicants next examined the effect of the inventivecompositions on the activation of NF-κB induced by cigarette smokecondensate in non-small lung adenocarcinoma H1299 cells. DNA-bindingassay (EMSA) results showed that the inventive compositions suppressedthe NF-κB activation induced by cigarette smoke condensate, as shown inFIG. 14(C). These results suggest that the inventive compositions actedat a step in the NF-κB activation pathway that is common to TNF andcigarette smoke condensate.

Example 11 Western Blot Analysis of TNF-Induced Gene Expression

To determine the effect of the inventive compositions on TNF-inducedexpression of COX-2, VEGF, ICAM-1, MMP-9, cIAP-1/2, survivin, Bfl-1/A1,Bcl-2, Bclx_(L), cFLIP, TRAF1, and XIAP in whole-cell extracts oftreated cells (2×10⁶ cells/ml), 30 μg of protein was resolved onSDS-PAGE and probed by Western blot with specific antibodies as permanufacturer's recommended protocol. The blots were washed, exposed toHRP-conjugated secondary antibodies for 1 h, and finally detected by ECLreagent (Amersham Pharmacia Biotechnology, Piscataway, N.J.).

The inventive compositions inhibit TNF-induced NF-κB dependent geneproducts involved in the proliferation and metastasis of tumor cells.Applicants also investigated whether the inventive compositions canmodulate NF-κB dependent gene products involved in the proliferation andmetastasis of tumor cells. TNF has been shown to induce COX-2, MMP-9,ICAM-1, and VEGF, all of which have NF-κB binding sites in theirpromoters. As shown in FIG. 15(A), TNF treatment induced the expressionof COX-2, VEGF, ICAM-1, and MMP-9 gene products and the inventivecompositions abolished the expression.

KBM-5 cells (2×10⁶ cells/ml) were incubated with 1 mg/ml of theinventive compositions for 1 h and then treated with 1 nM TNF for theindicated times. Whole-cell extracts were prepared and subjected toWestern blot analysis using the indicated antibodies, as shown in FIG.15(B).

The inventive compositions inhibit TNF-induced NF-κB dependentanti-apoptotic gene products. NF-κB upregulates the expression of theantiapoptotic proteins IAP1, IAP2, survivin, Bfl-1/A1, Bcl-2, Bcl-XL,cFLIP, TRAF1, and XIAP. Applicants next investigated whether theinventive compositions affects the expression of these gene products.Applicants found that the inventive compositions inhibits theTNF-induced as well as the basal expression of all of these proteins, asshown in FIG. 15(B).

Example 12 Evidence of G₂/M Arrest of PC3 Cells Mediated by theInventive Compositions

As shown in FIG. 16, treatment of PC3 cells with the inventivecompositions produces concentration-dependent inhibition of the cellcycle. The herbal product produces a clear G₂/M accumulation of cellsthat is evident even at concentrations as low as 0.28 ul/ml. At higherconcentrations, the G₂/M phase is more evident as is a sub G₀ peakindicative of apoptosis. Concomitant with the rise in numbers of cellsin G₂/M there is a progressive concentration-dependent fall of cells inthe G1 portion of the cell cycle. Only 10% of control cells areroutinely present at the G₂/M phase, while treatment with 0.57 ul/ml ofthe inventive compositions results in greater than 40% of cells in aG₂/M block.

As shown in FIG. 16A, the upper left plot demonstrates a normal cellcycle distribution of PC3 cells. The G2/M percentage of cells is small(small dark shaded peak to the right in the figure. In the second plot(upper right) PC3 cells have been treated with 0.28 ul of the inventivecompositions/ml tissue culture media. The size of the G2/M peakincreases. The plot in the lower left (0.57 ul of the inventivecompositions/ml) and in the lower right (1.14 ul/ml) show an increasingcell cycle block due to increased concentration of the inventivecompositions.

As shown in FIG. 16B, the data from the flow cytometry analyses in FIG.16A are replotted in the histogram here in FIG. 16B. As seen in thisfigure there is a clear concentration dependent increase in thepercentage of cells in the G2/M phases of the cell cycle while this isan associated decrease in the cells in the G1 phase of the cell cycle.

Example 13 Flow Cytometry Analyses of Apoptosis Mediated by theInventive Compositions

As shown in FIG. 17, concentrations of the inventive compositions thatproduced only low levels of G₂/M block were found to be associated withinduction of early apoptosis as evidenced by staining of cells withannexin V and phosphatidylinositol (PI). Even at the lowestconcentration examined, 0.28 ul/ml, clear evidence of annexin V bindingto inverted membrane phosphatidylserine is evident by flow cytometry ofthe inventive compositions treated PC3 cells. Further evidence of themulti-herb mediated production of apoptosis is in the concentrationresponse of propidium iodide binding to nucleic acids in later stages ofcell death following the breach of membrane integrity.

As shown in FIG. 17A, PC3 cells were treated with the inventivecompositions at indicated concentrations for 24 hr. Cells were thenstained with Annexin V and PI solution. Apoptosis was measured by flowcytometry analyses (FACS). Even at a low concentration of 0.28 ul/mlthere is a clear indication of early apoptosis. At concentrations inexcess of 1.1 ul/ml most cells have died and appear in a late apoptosis(cell death) stage.

As shown in FIG. 17B, data from flow cytometry analyses of the inventivecompositions treated PC3 cells (FIG. 17A) is depicted in a histogram inFIG. 17B. There is a clear concentration-dependent increase in PIstaining which is indicative of cell membrane alteration, a hallmark ofearly apoptosis. Annexin V staining even at the lowest concentration ofthe inventive compositions indicates DNA staining associated withapoptosis. The percentage of cells stained with Annexin V declines withincreasing concentration of the inventive compositions due to celldeath.

Example 14 Changes in Eicosanoid Metabolism Mediated by the InventiveCompositions

As shown in FIGS. 18A-18D, treatment of cloned human COX-1, COX-2 and5-LOX enzymes produces a concentration dependent inhibition of formationof respective eicosanoid products, PGE₂ and 5-HETE, with formation of5-LOX more potently inhibited than either COX-1 or COX-2 (data notshown). Cellular changes in eicosanoid metabolism due to incubation withthe inventive compositions were then examined. As seen in FIGS.18(A)-(D), treatment of PC3 cells with the inventive compositionsproduced a decrease in formation of PGE₂ (FIG. 18A) and 5-HETE (FIG.18B). More impressive, however, was the cellular decline in 12-HETElevels presumably resulting from inhibition of 12-LOX activity.Concentrations of the inventive compositions as low as 0.25 ul/minproduced a significant reduction in 12-HETE levels compared to untreatedcontrol cells. The highest concentration of the inventive compositionsexamined (1 ul/ml) resulted in an approximate 80% reduction in 12-HETElevels within PC3 cells (FIG. 18C).

The treatment of PC3 cells with the inventive compositions also produceda clear decrease in cellular content of 5-LOX protein as evidenced byWestern blot analysis (FIG. 18D). A decline in 12-LOX protein contentwas also evident at concentrations of the inventive compositions higherthan 0.25 ul/ml. The increase in COX-2 protein due to incubation of PC3cells with higher levels of the inventive compositions is reminiscent ofelevations of this enzyme due to treatment of cells with otheranti-inflammatory agents such as celecoxib.

The data in Table 5 were derived from phosphoprotein analyses of PC3cell lysates after treatment with nontoxic concentrations of either theinventive compositions or baicalein, a component of Scutellaria.Proteins were separated using gel electrophoresis and then the gel wasprobed with monoclonal antibodies to the proteins. The antibodies coulddetect differences in phosphorylation status of the proteins. Gels werethen quantified using a densitometer. Only relative changes (althoughsignificant) are indicated in the table above. There are manysimilarities in the changes in phosphorylation status signaling similarup regulation or down regulation of proteins involved in cell cycleblock (e.g. cyclin D1, cyclin D3 and pRB) or proteins associated withapoptosis (e.g. surviving, Bcl-X_(L) and Bcl-2). Both the inventivecompositions and baicalein also inhibit 12-LOX activity. However, notall the changes in phosphorylation status of these important proteins isidentical after treatment with either the inventive compositions orbaicalein, suggesting that the mechanism of action of the inventivecompositions is complex and is not due to its content of baicaleinalone.

TABLE 5 Activity or content Treatment with the measured in PC3 cellsinventive compositions Baicalein treatment Cytotoxicity Yes Yes Cellcycle block G2/M Yes 12-LOX activity ↓ ↓ pRb content ↓ ↓ Rb content ↑ ↑Cyclin D1 ↓ ↓ Cyclin D3 ↓ ↓ p21 No change Strong ↓ P27 No change Strong↓ Bcl-X_(L) ↓ ↓ Bax No change ↑ Bcl-2 ↓ ↓ pAKT No change ↓ survivin ↓ ↓↓ indicates reduction, inhibition, or downregulation ↑ indicatesenhancement, activation, or upregulation

Example 15 Decline in Phosphorylation of Rb Protein Mediated by theInventive Compositions

As shown in FIG. 19A, there is a concentration dependent inhibition ofRb phosphorylation. Concentrations as low as 0.25 ul/ml significantlyreduced pRb compared to controls while the amount of Rb increased as thepRb decreased. The decrease in phosphorylation of Rb protein occurred atconcentrations of the inventive compositions also associated with G₂/Mcell cycle block and early apoptosis. Quantitation of the Western blotdata is presented in FIG. 19B.

Shown in FIG. 19A is a Western blot of retinoblastoma protein as bothunphosphorylated (Rb) and phosphorylated (pRb) proteins. The bottom band(β-actin) is shown only as a loading control. The data are derived fromPC3 cells treated with the inventive compositions at the concentrationsshown for a 24 hr period of time. Cells were then lysed and proteinsseparated on a gel. The presence of Rb or pRb proteins was determinedwith use of specific monoclonal antibodies. The data are also shown inthe bottom figure after scanning the gels in a photometric scanningdevice to permit quantification of the density of the bands. The datashow that even at a concentration of 0.25 ul/ml of the inventivecompositions there is a clear suppression of pRb (phosphorylated form)and an increase in Rb (nonphosphorylated form).

The decrease in Rb phosphorylation occurred at multiple amino acid sitesincluding T356 (59% decrease relative to control), 5807 (62% ↓), T821(62% ↓) and T826 (78% ↓; other data not shown) resulting from incubationof PC3 cells with the inventive compositions at a concentration of 2ul/ml for 24 hr.

TABLE 6 Protein phosphorylation screening data from PC3 cells treatedwith the inventive compositions^(a) Change (%) in treated samplerelative to Protein Phosphorylation site controls Rb S807 −62 Rb T356−59 Rb T821 −62 Rb T826 −78 STAT1 S727 −38 Fos T232 +383 HspB1 S82 +421p21 S144/S141/S154 +136 ^(a)Abbreviations are as follows: PC3, androgeninsensitive human prostate cancer; Rb, retinoblastoma protein; STAT1,signal transducer and activator of transcription 1; Fos, Fos-c FBJmurine osteosarcoma oncoprotein-related transcription factor; HspB1,heat shock 27 kDa protein; p21, p21-activated protein serine kinase.

Example 16 Ability of 12-HETE to Block Inhibition of PC3 Proliferationby the Inventive Compositions

As shown in FIGS. 20A-20C, addition of PGE₂ or 5-HETE to PC3 cellstreated with growth inhibitory concentrations of the inventivecompositions failed to block cellular proliferation. In contrast, FIG.20A shows that 12-HETE added to cells treated with the inventivecompositions (1 ul/ml; a concentration that produced potent induction ofapoptosis and G₂/M arrest) resulted in a near doubling of cellproliferation although not back to control untreated cell levels.

Addition of 12-HETE (designated as 12-H) also resulted in a block of theability of the inventive compositions to reverse the phosphorylatedstatus of RB protein in PC3 cells (FIG. 20B); neither PGE2 nor 5-HETE(designated as 5H) had any effect on the ability of the inventivecompositions to alter pRb status. Finally, 12-HETE added to PC3 cellsalso provided partial reversal of the inventive compositions mediatedG2/M block thus pointing out the importance of this particulareicosanoid in inhibition of PC3 cell proliferation mediated by theinventive compositions.

Thus, as shown in FIG. 20A in an “add-back” experiment, PC3 cells weretreated with the inventive compositions at the indicated concentrationswhich resulted in a concentration dependent inhibition of cellproliferation (relative fluorescence indicates numbers of cells).Addition of 12-HETE, the product of 12-lipoxygenase, resulted in amodest increase in PC3 cell growth. However, addition of 12-HETE tocells treated with the inventive compositions largely blocked tumor cellproliferation inhibition mediated by the inventive compositions.

As shown in FIG. 20B, the inventive compositions produced aconcentration dependent decline in expression of pRb. Adding either5-HETE (5-H) or PGE2 did not block this effect. Addition of the 12-LOXproduct, 12-HETE, however, partially returned the production of pRbexpression. Taken together these data show that the inhibition of 12-LOXmediated by the inventive compositions is important in that 12-HETE canblock anti-proliferative activity and the ability of the inventivecompositions to restore expression of the tumor suppressor protein Rb.

REFERENCES

The following literature references are believed to useful to anunderstanding of the inventive subject matter in the context of itsplace in the relevant art. Citation here is not to be construed as anassertion or admission that any reference cited is material topatentability of the inventive subject matter. Applicants will properlydisclose information material to patentability in an InformationDisclosure Statement. The content of each reference is herebyincorporated in their entirety.

-   1. Singh, S, and Aggarwal, B. B. (1995) Activation of transcription    factor NF-kappa B is suppressed by curcumin (diferuloylmethane)    [corrected]. J. Biol. Chem., 270, 24995-25000.-   2. Aggarwal, S., Ichikawa, H., Takada, Y., Sandur, S. K., Shishodia,    S, and Aggarwal, B. B. (2006) Curcumin (diferuloylmethane)    down-regulates expression of cell proliferation and antiapoptotic    and metastatic gene products through suppression of I kappa B alpha    kinase and Akt activation. Mol. Pharmacol., 69, 195-206.-   3. Plummer, S. M., Holloway, K. A., Manson, M. M., Munks, R. J.,    Kaptein, A., Farrow, S, and Howells, L. (1999) Inhibition of    cyclooxygenase 2 expression in colon cells by the chemopreventive    agent curcumin involves inhibition of NF kappa B activation via the    NIK/IKK signalling complex. Oncogene, 18, 6013-6020.-   4. Paschka, A. G., Butler, R. and Young, C. Y. (1998) Induction of    apoptosis in prostate cancer cell lines by the green tea component,    (−)-epigallocatechin-3-gallate. Cancer Lett, 130, 1-7.-   5. Kim, D. S., Kim, H. R., Woo, E. R., Hong, S. T., Chae, H. J. and    Chae, S. W. (2005) Inhibitory effects of rosmarinic acid on    adriamycin-induced apoptosis in H9c2 cardiac muscle cells by    inhibiting reactive oxygen species and the activations of c-Jun    N-terminal kinase and extracellular signal-regulated kinase.    Biochem. Pharmacol., 70, 1066-1078.-   6. Huang, S. S, and Zheng, R. L. (2005) Rosmarinic acid inhibits    angiogenesis and its mechanism of action in vitro. Cancer Lett.-   7. Shishodia, S., Majumdar, S., Banerjee, S, and    Aggarwal, B. B. (2003) Ursolic acid inhibits nuclear factor-kappaB    activation induced by carcinogenic agents through suppression of I    kappa B alpha kinase and p65 phosphorylation: correlation with    down-regulation of cyclooxygenase 2, matrix metalloproteinase 9, and    cyclin D1. Cancer Res., 63, 4375-4383.-   8. Choi, Y. H., Baek, J. H., Yoo, M. A., Chung, H. Y., Kim, N. D.    and Kim, K. W. (2000) Induction of apoptosis by ursolic acid through    activation of caspases and downregulation of c-IAPs in human    prostate epithelial cells. Int. J. Oncol., 17, 565-571.-   9. Kim, S. O., Kundu, J. K., Shin, Y. K., Park, J. H., Cho, M. H.,    Kim, T. Y. and Surh, Y. J. (2005) [6]-Gingerol inhibits COX-2    expression by blocking the activation of p38 MAP kinase and    NF-kappaB in phorbol ester-stimulated mouse skin. Oncogene, 24,    2558-2567.-   10. Atsumi, T., Murakami, Y., Shibuya, K., Tonosaki, K. and    Fujisawa, S. (2005) Induction of cytotoxicity and apoptosis and    inhibition of cyclooxygenase-2 gene expression, by curcumin and its    analog, alpha-diisoeugenol. Anticancer Res., 25, 4029-4036.-   11. Tjendraputra, E., Tran, V. H., Liu-Brennan, D.,    Roufogalis, B. D. and Duke, C. C. (2001) Effect of ginger    constituents and synthetic analogues on cyclooxygenase-2 enzyme in    intact cells. Bioorg. Chem., 29, 156-163.-   12. Manna, S. K., Mukhopadhyay, A. and Aggarwal, B. B. (2000)    Resveratrol suppresses TNF-induced activation of nuclear    transcription factors NF-kappa B, activator protein-1, and    apoptosis: potential role of reactive oxygen intermediates and lipid    peroxidation. J. Immunol., 164, 6509-6519.-   13. Fukuda, K., Hibiya, Y., Mutoh, M., Koshiji, M., Akao, S, and    Fujiwara, H. (1999) Inhibition by berberine of cyclooxygenase-2    transcriptional activity in human colon cancer cells. J.    Ethnopharmacol., 66, 227-233.-   14. Kelm, M. A., Nair, M. G., Strasburg, G. M. and    DeWitt, D. L. (2000) Antioxidant and cyclooxygenase inhibitory    phenolic compounds from Ocimum sanctum Linn. Phytomedicine, 7, 7-13.-   15. Bemis, D. L., Capodice, J. L., Anastasiadis, A. G., Katz, A. E.    and Buttyan, R. (2005) Zyflamend, a unique herbal preparation with    nonselective COX inhibitory activity, induces apoptosis of prostate    cancer cells that lack COX-2 expression. Nutr. Cancer., 52, 202-212.-   16. Aggarwal, B. B. (2004) Nuclear factor-kappaB: the enemy within.    Cancer Cell, 6, 203-208.-   17. Anto, R. J., Mukhopadhyay, A., Shishodia, S., Gairola, C. G. and    Aggarwal, B. B. (2002) Cigarette smoke condensate activates nuclear    transcription factor-kappaB through phosphorylation and degradation    of IkappaB(alpha): correlation with induction of cyclooxygenase-2.    Carcinogenesis, 23, 1511-1518.-   18. Bharti, A. C., Takada, Y., Shishodia, S, and    Aggarwal, B. B. (2004) Evidence that receptor activator of nuclear    factor (NF)-kappaB ligand can suppress cell proliferation and induce    apoptosis through activation of a NF-kappaB independent and    TRAF6-dependent mechanism. J. Biol. Chem., 279, 6065-6076.-   19. Takada, Y., Ichikawa, H., Badmaev, V. and Aggarwal, B. B. (2006)    Acetyl-1′-ketobeta-boswellic acid potentiates apoptosis, inhibits    invasion, and abolishes osteoclastogenesis by suppressing NF-kappaB    and NF-kappaB-regulated gene expression. J. Immunol., 176,    3127-3140.-   20. Chaturvedi, M. M., Mukhopadhyay, A. and Aggarwal, B. B. (2000)    Assay for redox-sensitive transcription factor. Methods Enzymol.,    319, 585-602.-   21. Abu-Amer, Y. and Tondravi, M. M. (1997) NF-kappaB and bone: the    breaking point. Nat. Med., 3, 1189-1190.-   22. Liotta, L. A., Thorgeirsson, U. P. and Garbisa, S. (1982) Role    of collagenases in tumor cell invasion. Cancer Metastasis Rev., 1,    277-288.-   23. Van Antwerp, D. J., Martin, S. J., Kafri, T., Green, D. R. and    Verma, I. M. (1996) Suppression of TNF-alpha-induced apoptosis by    NF-kappaB. Science, 274, 787-789.-   24. Wang, C. Y., Mayo, M. W. and Baldwin, A. S., Jr. (1996) TNF- and    cancer therapy induced apoptosis: potentiation by inhibition of    NF-kappaB. Science, 274, 784-787.-   25. Yamamoto, K., Arakawa, T., Ueda, N. and Yamamoto, S. (1995)    Transcriptional roles of nuclear factor kappa B and nuclear    factor-interleukin-6 in the tumor necrosis factor alpha-dependent    induction of cyclooxygenase-2 in MC3T3-E1 cells. J. Biol. Chem.,    270, 31315-31320.-   26. Esteve, P. O., Chicoine, E., Robledo, O., Aoudjit, F.,    Descoteaux, A., Potworowski, E. F. and St-Pierre, Y. (2002) Protein    kinase C-zeta regulates transcription of the matrix    metalloproteinase-9 gene induced by IL-1 and TNF-alpha in glioma    cells via NF-kappa B. J. Biol. Chem., 277, 35150-35155.-   27. van de Stolpe, A., Caldenhoven, E., Stade, B. G., Koenderman,    L., Raaijmakers, J. A., Johnson, J. P. and van der    Saag, P. T. (1994) 12-O-tetradecanoylphorbol-13-acetate and tumor    necrosis factor alpha-mediated induction of intercellular adhesion    molecule-1 is inhibited by dexamethasone. Functional analysis of the    human intercellular adhesion molecular-1 promoter. J. Biol. Chem.,    269, 6185-6192.-   28. Zhu, L., Fukuda, S., Cordis, G., Das, D. K. and    Maulik, N. (2001) Anti-apoptotic protein survivin plays a    significant role in tubular morphogenesis of human coronary    arteriolar endothelial cells by hypoxic preconditioning. FEBS Lett.,    508, 369-374.-   29. Chu, Z. L., McKinsey, T. A., Liu, L., Gentry, J. J.,    Malim, M. H. and Ballard, D. W. (1997) Suppression of tumor necrosis    factor-induced cell death by inhibitor of apoptosis c-IAP2 is under    NF-kappaB control. Proc. Natl. Acad. Sci. USA., 94, 10057-10062.-   30. You, M., Ku, P. T., Hrdlickova, R. and Bose, H. R., Jr. (1997)    ch-IAP1, a member of the inhibitor-of-apoptosis protein family, is a    mediator of the antiapoptotic activity of the v-Rel oncoprotein.    Mol. Cell. Biol., 17, 7328-7341.-   31. Catz, S. D. and Johnson, J. L. (2001) Transcriptional regulation    of bcl-2 by nuclear factor kappa B and its significance in prostate    cancer. Oncogene, 20, 7342-7351.-   32. Stehlik, C., de Martin, R., Kumabashiri, I., Schmid, J. A.,    Binder, B. R. and Lipp, J. (1998) Nuclear factor    (NF)-kappaB-regulated X-chromosome-linked iap gene expression    protects endothelial cells from tumor necrosis factor alpha-induced    apoptosis. J. Exp. Med., 188, 211-216.-   33. Tamatani, M., Che, Y. H., Matsuzaki, H., Ogawa, S., Okado, H.,    Miyake, S., Mizuno, T. and Tohyama, M. (1999) Tumor necrosis factor    induces Bcl-2 and Bclx expression through NFkappaB activation in    primary hippocampal neurons. J. Biol. Chem., 274, 8531-8538.-   34. Schwenzer, R., Siemienski, K., Liptay, S., Schubert, G., Peters,    N., Scheurich, P., Schmid, R. M. and Wajant, H. (1999) The human    tumor necrosis factor (TNF) receptor-associated factor 1 gene    (TRAF1) is up-regulated by cytokines of the TNF ligand family and    modulates TNF-induced activation of NF-kappaB and c-Jun N-terminal    kinase. J. Biol. Chem., 274, 19368-19374.-   35. Kreuz, S., Siegmund, D., Scheurich, P. and Wajant, H. (2001)    NF-kappaB inducers upregulate cFLIP, a cycloheximide-sensitive    inhibitor of death receptor signaling. Mol. Cell. Biol., 21,    3964-3973.-   36. Shishodia, S, and Aggarwal, B. B. (2004) Nuclear factor-kappaB    activation mediates cellular transformation, proliferation, invasion    angiogenesis and metastasis of cancer. Cancer Treat Res., 119,    139-173.-   37. Takada, Y., Singh, S, and Aggarwal, B. B. (2004) Identification    of a p65 peptide that selectively inhibits NF-kappa B activation    induced by various inflammatory stimuli and its role in    down-regulation of NF-kappaB-mediated gene expression and    up-regulation of apoptosis. J. Biol. Chem., 279, 15096-15104.

The inventive subject matter being thus described, it will be obviousthat the same may be modified or varied in many ways. Such modificationsand variations are not to be regarded as a departure from the spirit andscope of the inventive subject matter and all such modifications andvariations are intended to be included within the scope of the followingclaims.

1. A method for modulating an eicosanoid metabolic process in cells ofan animal in need thereof, comprising: administering to the animal anamount of a composition comprising therapeutically effective amounts ofsupercritical extracts of rosemary, turmeric, oregano and ginger; andtherapeutically effective amounts of hydroalcoholic extracts of holybasil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea,huzhang, Chinese goldthread, and barberry; and whereby the administeredcomposition inhibits lipoxygenase activity in the animal and increasesthe concentration of 13-HODE in the animal.
 2. The method of claim 1,wherein the liopoxygenase activity inhibited in the animal is 5-LOXactivity.
 3. The method of claim 2, wherein the 5-LOX activity and12-LOX activity in the animal is inhibited simultaneously with anincrease in concentration of 13-HODE in the animal.
 4. The method ofclaim 1, wherein the liopoxygenase activity inhibited in the animal is12-LOX activity.
 5. The method of claim 1, wherein the wherein theliopoxygenase activity inhibited in the animal is both 5-LOX activityand 12-LOX activity.
 6. A method of delivering 13-S-HODE to an animal inneed thereof, comprising administering to the animal a compositioncomprising therapeutically effective amounts of supercritical extractsof rosemary, turmeric, oregano and ginger; and therapeutically effectiveamounts of hydroalcoholic extracts of holy basil, ginger, turmeric,Scutellaria baicalensis, rosemary, green tea, huzhang, Chinesegoldthread, and barberry.
 7. A method of for inhibiting both 5-LOXactivity and 12-LOX activity in an animal in need thereof, comprisingadministering to the animal a composition comprising therapeuticallyeffective amounts of supercritical extracts of rosemary, turmeric,oregano and ginger; and therapeutically effective amounts ofhydroalcoholic extracts of holy basil, ginger, turmeric, Scutellariabaicalensis, rosemary, green tea, huzhang, Chinese goldthread, andbarberry.